Novel genes, compositions, kits and methods for identification, assessment, prevention, and therapy of cervical cancer

ABSTRACT

The invention relates to newly discovered nucleic acid molecules and proteins associated with cervical cancer including pre-malignant conditions such as dysplasia. Compositions, kits, and methods for detecting, characterizing, preventing, and treating human cervical cancers are provided.

RELATED APPLICATIONS

[0001] The present application claims priority to U.S. provisionalpatent application serial No. 60/298,159, filed on Jun. 13, 2001, U.S.provisional patent application serial No. 60/298,155, filed on Jun. 13,2001, and U.S. provisional patent application serial No. 60/335,936,filed on Nov. 14, 2001, all of which are expressly incorporated byreference.

FIELD OF THE INVENTION

[0002] The field of the invention is cervical cancer, includingdiagnosis, characterization, management, and therapy of cervical cancer.

BACKGROUND OF THE INVENTION

[0003] The increased number of cancer cases reported in the UnitedStates, and, indeed, around the world, is a major concern. Currentlythere are only a handful of treatments available for specific types ofcancer, and these provide no absolute guarantee of success. In order tobe most effective, these treatments require not only an early detectionof the malignancy, but a reliable assessment of the severity of themalignancy.

[0004] Cancer of the cervix is one of the most common malignancies inwomen and remains a significant public health problem throughout theworld. In the United States alone, invasive cervical cancer accounts forapproximately 19% of all gynecological cancers. In 1996, it wasestimated that there were 14,700 newly diagnosed cases and 4900 deathsattributed to this disease (American Cancer Society, Cancer Facts &FIGS. 1996, Atlanta, Ga.: American Cancer Society, 1996). In manydeveloping countries, where mass screening programs are not widelyavailable, the clinical problem is more serious. Worldwide, the numberof new cases is estimated to be 471,000 with a four-year survival rateof only 40% (Munoz et al., 1989, Epidemiology of Cervical Cancer In:“Human Papillomavirus”, New York, Oxford Press, pp 9-39; NationalInstitutes of Health, Consensus Development Conference Statement onCervical Cancer, Apr.1-3, 1996).

[0005] The precursor to cervical cancer is dysplasia, also known in theart as cervical intraepithelial neoplasia (CIN) or squamousintraepithelial lesions (SIL). While it is not understood how normalcells become transformed, the concept of a continuous spectrum ofhistopathological change from normal, stratified epithelium through CINto invasive cancer has been widely accepted for many years. A large bodyof epidemiological and molecular biological evidence has establishedhuman papillomavirus (HPV) infection as a causative factor in cervicalcancer. HPV is found in 85% or more of squamous cell invasive lesions,which represent the most common histologic type seen in cervicalcarcinoma. Additional cofactors have also been identified, includingoncogenes that have been activated by point mutations and chromosomaltranslocations or deletions.

[0006] In light of this, cervical cancer remains a highly preventableform of cancer when pre-invasive lesions are detected early. Cytologicalexamination of Papanicolaou-stained cervical smears (also referred to asPap smears) is currently the principle method for detecting cervicalcancer. Not surprisingly, the effectiveness of Pap smear screeningvaries depending not only upon the quality of the sample being used, butalso upon subjective parameters that are inherent to the analysis. Inaddition, despite the historical success of the test, concerns havearisen regarding its ability to reliably predict the behavior of somepre-invasive lesions (Ostor et al., 1993, Int. J Gynecol. Pathol. 12:186-192; and Genest et al., 1993, Human Pathol. 24: 730-736).

SUMMARY OF THE INVENTION

[0007] The invention relates to cancer markers (hereinafter “markers” or“markers of the inventions”), which are listed in Table 1. The inventionprovides nucleic acids and proteins that are encoded by or correspond tothe markers (hereinafter “marker nucleic acids” and “marker proteins,”respectively). Table 1 provides the sequence identifiers of thesequences of such marker nucleic acids and proteins listed in theaccompanying Sequence Listing. The invention further providesantibodies, antibody derivatives and antibody fragments which bindspecifically with such proteins and/or fragments of the proteins.

[0008] The invention also relates to various methods, reagents and kitsfor diagnosing, staging, prognosing, monitoring and treating cervicalcancer. “Cervical cancer ” as used herein includes carcinomas, (e.g.,carcinoma in situ, invasive carcinoma, metastatic carcinoma) andpre-malignant conditions, (e.g., dysplasia, including CIN or SIL). Inone embodiment, the invention provides a diagnostic method of assessingwhether a patient has cervical cancer or has higher than normal risk fordeveloping cervical cancer, comprising the steps of comparing the levelof expression of a marker of the invention in a patient sample and thenormal level of expression of the marker in a control, e.g., a samplefrom a patient without cervical cancer. A significantly higher level ofexpression of the marker in the patient sample as compared to the normallevel is an indication that the patient is afflicted with cervicalcancer or has higher than normal risk for developing cervical cancer.

[0009] According to the invention, the markers are selected such thatthe positive predictive value of the methods of the invention is atleast about 10%, preferably about 25%, more preferably about 50% andmost preferably about 90%. Also preferred for use in the methods of theinvention are markers that are differentially expressed, as compared tonormal cervical cells, by at least two-fold in at least about 20%,morepreferably about 50% and most preferably about 75% of any of thefollowing conditions: stage 0 cervical cancer patients, stage I cervicalcancer patients, stage II cervical cancer patients, stage III cervicalcancer patients, stage IV cervical cancer patients, grade I cervicalcancer patients, grade II cervical cancer patients, grade III cervicalcancer patients, squamous cell (epidermoid) cervical cancer patients,cervical adenocarcinoma patients, cervical adenosquamous carcinomapatients, small-cell cervical carcinoma patients, malignant cervicalcancer patients, patients with primary carcinomas of the cervix,patients with primary malignant lymphomas of the cervix and patientswith secondary malignant lymphomas of the cervix, and all other types ofcancers, malignancies and transformations associated with the cervix.

[0010] In a preferred diagnostic method of assessing whether a patientis afflicted with cervical cancer (e.g., new detection (“screening”),detection of recurrence, reflex testing), the method comprisescomparing:

[0011] a) the level of expression of a marker of the invention in apatient sample, and

[0012] b) the normal level of expression of the marker in a controlnon-cervical cancer sample.

[0013] A significantly higher level of expression of the marker in thepatient sample as compared to the normal level is an indication that thepatient is afflicted with cervical cancer.

[0014] The invention also provides diagnostic methods for assessing theefficacy of a therapy for inhibiting cervical cancer in a patient. Suchmethods comprise comparing:

[0015] a) expression of a marker of the invention in a first sampleobtained from the patient prior to providing at least a portion of thetherapy to the patient, and

[0016] b) expression of the marker in a second sample obtained from thepatient following provision of the portion of the therapy.

[0017] A significantly lower level of expression of the marker in thesecond sample relative to that in the first sample is an indication thatthe therapy is efficacious for inhibiting cervical cancer in thepatient.

[0018] It will be appreciated that in these methods the “therapy” may beany therapy for treating cervical cancer including, but not limited to,chemotherapy, radiation therapy, surgical removal of tumor tissue, genetherapy and biologic therapy such as the administering of antibodies andchemokines. Thus, the methods of the invention may be used to evaluate apatient before, during and after therapy, for example, to evaluate thereduction in tumor burden.

[0019] In a preferred embodiment, the diagnostic methods are directed totherapy using a chemical or biologic agent. These methods comprisecomparing:

[0020] a) expression of a marker of the invention in a first sampleobtained from the patient and maintained in the presence of the chemicalor biologic agent, and

[0021] b) expression of the marker in a second sample obtained from thepatient and maintained in the absence of the agent.

[0022] A significantly lower level of expression of the marker in thesecond sample relative to that in the first sample is an indication thatthe agent is efficacious for inhibiting cervical cancer, in the patient.In one embodiment, the first and second samples can be portions of asingle sample obtained from the patient or portions of pooled samplesobtained from the patient.

[0023] The invention additionally provides a monitoring method forassessing the progression of cervical cancer in a patient, the methodcomprising:

[0024] a) detecting in a patient sample at a first time point, theexpression of a marker of the invention;

[0025] b) repeating step a) at a subsequent time point in time; and

[0026] c) comparing the level of expression detected in steps a) and b),and therefrom monitoring the progression of cervical cancer in thepatient.

[0027] A significantly higher level of expression of the marker in thesample at the subsequent time point from that of the sample at the firsttime point is an indication that the cervical cancer has progressed,whereas a significantly lower level of expression is an indication thatthe cervical cancer has regressed.

[0028] The invention further provides a diagnostic method fordetermining whether cervical cancer has metastasized or is likely tometastasize in the future, the method comprising comparing:

[0029] a) the level of expression of a marker of the invention in apatient sample, and

[0030] b) the normal level (or non-metastatic level) of expression ofthe marker in a control sample.

[0031] A significantly higher level of expression in the patient sampleas compared to the normal level (or non-metastatic level) is anindication that the cervical cancer has metastasized or is likely tometastasize in the future.

[0032] The invention moreover provides a test method for selecting acomposition for inhibiting cervical cancer in a patient. This methodcomprises the steps of:

[0033] a) obtaining a sample comprising cancer cells from the patient;

[0034] b) separately maintaining aliquots of the sample in the presenceof a plurality of test compositions;

[0035] c) comparing expression of a marker of the invention in each ofthe aliquots; and

[0036] d) selecting one of the test compositions which significantlyreduces the level of expression of the marker in the aliquot containingthat test composition, relative to the levels of expression of themarker in the presence of the other test compositions.

[0037] The invention additionally provides a test method of assessingthe cervical carcinogenic potential of a compound. This method comprisesthe steps of:

[0038] a) maintaining separate aliquots of cervical cells in thepresence and absence of the compound; and

[0039] b) comparing expression of a marker of the invention in each ofthe aliquots.

[0040] A significantly higher level of expression of the marker in thealiquot maintained in the presence of the compound, relative to that ofthe aliquot maintained in the absence of the compound, is an indicationthat the compound possesses cervical carcinogenic potential.

[0041] In addition, the invention further provides a method ofinhibiting cervical cancer in a patient. This method comprises the stepsof:

[0042] a) obtaining a sample comprising cancer cells from the patient;

[0043] b) separately maintaining aliquots of the sample in the presenceof a plurality of compositions;

[0044] c) comparing expression of a marker of the invention in each ofthe aliquots; and

[0045] d) administering to the patient at least one of the compositionswhich significantly lowers the level of expression of the marker in thealiquot containing that composition, relative to the levels ofexpression of the marker in the presence of the other compositions.

[0046] In the aforementioned methods, the samples or patient samplescomprise cells obtained from the patient. The cells may be found in acervical smear collected, for example, by a cervical brush. In anotherembodiment, the sample is a body fluid. Such fluids include, forexample, blood fluids, lymph, ascitic fluids, gynecological fluids,urine, and fluids collected by vaginal rinsing. In a further embodiment,the patient sample is in vivo.

[0047] According to the invention, the level of expression of a markerof the invention in a sample can be assessed, for example, by detectingthe presence in the sample of:

[0048] the corresponding marker protein (e.g., a protein having one ofthe sequences set forth as “SEQ ID NO (AAs)” in Table 1, or a fragmentof the protein (e.g. by using a reagent, such as an antibody, anantibody derivative, an antibody fragment or single-chain antibody,which binds specifically with the protein or protein fragment)

[0049] the corresponding marker nucleic acid (e.g. a nucleotidetranscript having one of the nucleic acid sequences set forth as “SEQ IDNO (nts)” in Table 1, or a complement thereof), or a fragment of thenucleic acid (e.g. by contacting transcribed polynucleotides obtainedfrom the sample with a substrate having affixed thereto one or morenucleic acids having the entire or a segment of the nucleic acidsequence of any of the SEQ ID NO (nts), or a complement thereof)

[0050] a metabolite which is produced directly (i.e., catalyzed) orindirectly by the corresponding marker protein.

[0051] According to the invention, any of the aforementioned methods maybe performed using a plurality (e.g. 2, 3, 5, or 10 or more) of cervicalcancer markers, including cervical cancer markers known in the art. Insuch methods, the level of expression in the sample of each of aplurality of markers, at least one of which is a marker of theinvention, is compared with the normal level of expression of each ofthe plurality of markers in samples of the same type obtained fromcontrol humans not afflicted with cervical cancer. A significantlyaltered (i.e., increased or decreased as specified in theabove-described methods using a single marker) level of expression inthe sample of one or more markers of the invention, or some combinationthereof, relative to that marker's corresponding normal or controllevel, is an indication that the patient is afflicted with cervicalcancer. For all of the aforementioned methods, the marker(s) arepreferably selected such that the positive predictive value of themethod is at least about 10%.

[0052] In a further aspect, the invention provides an antibody, anantibody derivative, or an antibody fragment, which binds specificallywith a marker protein (e.g., a protein having one of the amino acidsequences set forth in the Sequence Listing) or a fragment of theprotein. The invention also provides methods for making such antibody,antibody derivative, and antibody fragment. Such methods may compriseimmunizing a mammal with a protein or peptide comprising the entirety,or a segment of 10 or more amino acids, of a marker protein (e.g., aprotein having one of the amino acid sequences set forth in the SequenceListing), wherein the protein or peptide may be obtained from a cell orby chemical synthesis. The methods of the invention also encompassproducing monoclonal and single-chain antibodies, which would furthercomprise isolating splenocytes from the immunized mammal, fusing theisolated splenocytes with an immortalized cell line to form hybridomas,and screening individual hybridomas for those that produce an antibodythat binds specifically with a marker protein or a fragment of theprotein.

[0053] In another aspect, the invention relates to various diagnosticand test kits. In one embodiment, the invention provides a kit forassessing whether a patient is afflicted with cervical cancer. The kitcomprises a reagent for assessing expression of a marker of theinvention. In another embodiment, the invention provides a kit forassessing the suitability of a chemical or biologic agent for inhibitingcervical cancer in a patient. Such a kit comprises a reagent forassessing expression of a marker of the invention, and may also compriseone or more of such agents. In a further embodiment, the inventionprovides kits for assessing the presence of cervical cancer cells ortreating cervical cancers. Such kits comprise an antibody, an antibodyderivative, or an antibody fragment, which binds specifically with amarker protein, or a fragment of the protein. Such kits may alsocomprise a plurality of antibodies, antibody derivatives, or antibodyfragments wherein the plurality of such antibody agents bindsspecifically with a marker protein, or a fragment of the protein.

[0054] In an additional embodiment, the invention also provides a kitfor assessing the presence of cervical cancer cells, wherein the kitcomprises a nucleic acid probe that binds specifically with a markernucleic acid or a fragment of the nucleic acid. The kit may alsocomprise a plurality of probes, wherein each of the probes bindsspecifically with a marker nucleic acid, or a fragment of the nucleicacid.

[0055] In a further aspect, the invention relates to methods fortreating a patient afflicted with cervical cancer or at risk ofdeveloping cervical cancer. Such methods may comprise reducing theexpression and/or interfering with the biological function of a markerof the invention. In one embodiment, the method comprises providing tothe patient an antisense oligonucleotide or polynucleotide complementaryto a marker nucleic acid, or a segment thereof. For example, anantisense polynucleotide may be provided to the patient through thedelivery of a vector that expresses an anti-sense polynucleotide of amarker nucleic acid or a fragment thereof. In another embodiment, themethod comprises providing to the patient an antibody, an antibodyderivative, or antibody fragment, which binds specifically with a markerprotein or a fragment of the protein. In a preferred embodiment, theantibody, antibody derivative or antibody fragment binds specificallywith a protein having one of the amino acid sequences set forth in theSequence Listing, or a fragment of the protein.

[0056] It will be appreciated that the methods and kits of the presentinvention may also include known cancer markers including known cervicalcancer markers. It will further be appreciated that the methods and kitsmay be used to identify cancers other than cervical cancer.

DETAILED DESCRIPTION OF THE INVENTION

[0057] The invention relates to newly discovered cancer markersassociated with the cancerous state of cervical cells. It has beendiscovered that the higher than normal level of expression of any ofthese markers or combination of these markers correlates with thepresence of cervical cancer including pre-malignant conditions such asdysplasia, in a patient. Methods are provided for detecting the presenceof cervical cancer in a sample, the absence of cervical cancer in asample, the stage of a cervical cancer, and other characteristics ofcervical cancer that are relevant to prevention, diagnosis,characterization, and therapy of cervical cancer in a patient. Methodsof treating cervical cancer are also provided.

[0058] Table 1 lists the markers of the invention which areover-expressed in cervical cancer cells compared to normal (i.e.,non-cancerous) cervical cells and comprises markers listed in Tables 2and 3. Table 2 lists newly-identified nucleotide and amino acidsequences. Table 3 lists newly-identified nucleotide sequences. Tables1-3 provide the sequence listing identifiers of the cDNA sequence of anucleotide transcript and the amino acid sequence of a protein encodedby or corresponding to each marker, as well as the location of theprotein coding sequence within the CDNA sequence. TABLE 1 SEQ ID NO SEQID Marker Gene Name (nts) NO (AAs) CDS M661 AKAP9: A kinase 1 2  223 . .. 11946 (PRKA) anchor protein (yotiao) 9, variant 1 M662 AKAP9: A kinase3 4  223 . . . 11922 (PRKA) anchor protein (yotiao) 9, variant 2 M663AKAP9: A kinase 5 6  223 . . . 12000 (PRKA) anchor protein (yotiao) 9,variant 3 M664 AKAP9: A kinase 7 8  223 . . . 11976 (PRKA) anchorprotein (yotiao) 9, variant 4 M1 APOL1: Apolipopro- 9 10  213 . . . 1364tein L-I mNA, splice variant A, major form M2 APOL1: Apolipopro- 11 12 274 . . . 1518 tein L-I mNA, splice variant B, minor form M3 APOL3:apolipopro- 13 14  418 . . . 1413 tein L, 3; TNF- inducible proteinCG12-1 OV3 AQP5: Aquaporin 5 15 16  519 . . . 1316 M4 BC001980: clone 1718  157 . . . 225 MGC:5618 M5 BST2: Bone marrow 19 20  10 . . . 552stromal cell antigen 2 M6 BTEB1: basic tran- 21 22 1265 . . . 1999scription element binding protein 1 M665 CD74: CD74 antigen 23 24   8 .. . 706 (invariant polypeptide of major histocompati- bility complex,class II antigen-associated) M7 CDC20: CDC20 cell 25 26  45 . . . 1544cycle protein M8 CDKN2C: cyclin- 27 28 1216 . . . 1722 dependent kinaseinhibitor 2C, p18 M9 CKTSF1B1: (cysteine 29 30  45 . . . 1544 knotsuperfamily 1, BMP antagonist 1), gremlin M10 CLDN1: claudin 1 31 32 221 . . . 856 M11 CLIC4: chloride 33 34  198 . . . 959 intracellularchannel 4 M12 COL1A1: collagen, 35 36  120 . . . 4514 type I, alpha 1M13 COL1A2: collagen, 37 38  140 . . . 4240 type I, alpha 2 M14 COL8A1:collagen, 39 40   1 . . . 2235 type VIII, alpha 1 M15 COPA: coatomer 4142  467 . . . 4141 protein complex, subunit alpha M16 CRIP1:cysteine-rich 43 44   1 . . . 234 protein 1 (intestinal) M17 CTGF:connective 45 46  146 . . . 1195 tissue growth factor M18 DOC:downregulated 47 48  135 . . . 2393 in ovarian cancer 1 M19 EFNA1:ephrin-A1 49 50  74 . . . 691 M481 EPPK1: epiplakin 1 51 52  89 . . .15286 M20 FLJ11350: hypothet- 53 54  106 . . . 1047 ical proteinFLJ11350 M21 FLJ13809: hypothet- 55 56  64 . . . 1593 ical proteinFLJ13809 M22 FLJ20500: hypothet- 57 58  198 . . . 896 ical proteinFLJ20500 M23 FLJ23399: hypothet- 59 60  283 . . . 1770 ical proteinFLJ23399 M24 FN1: Fibronectin 1, 61 62  <1 . . . 2384 variant 1 M25 FN1:Fibronectin 1, 63 64  <1 . . . 6988 variant 2 M482 FOSL2: FOS-like 65 66 324 . . . 1304 antigen 2, variant 1 M483 FOSL2: FOS-like 67 66  324 . .. 1304 antigen 2, variant 2 M484 FSHPRH1: FSH 68 69  270 . . . 2540primary response (LRPR1, rat) homolog 1 M26 FY: Duffy blood group 70 71 495 . . . 1511 M485 G1P3:interferon, 72 73  108 . . . 500alpha-inducible protein (clone IFI-6-16) M486 GW112: GW112 74 75  509 .. . 1072 protein M27 HSKERUV: clone 76 77  <1 . . . 801 266, Humanradiated keratinocyte mRNA 266 (keratin-related protein) M28 HSPC121:butyrate- 78 79  150 . . . 1271 induced transcript 1 M29 HUMCLPB:Coactosin 80 81  150 . . . 576 like protein M487 hypothetical protein 8283  58 . . . 8163 M30 IFI27: (interferon, 84 85  55 . . . 423alpha-inducible protein 27 OV31 IFI30: interferon, 86 87  41 . . . 952gamma-inducible protein 30 M31 IFITM2: interferon 88 89  280 . . . 678induced transmem- brane protein 2 (1-8D) M32 IGFBP-3: insulin-like 90 91 133 . . . 1009 growth factor binding protein 3 M33 IL8RA: interleukin 892 93  75 . . . 374 M34 INHBA: Inhibin, 94 95  86 . . . 1366 beta-1 M488ITGA3: integrin, alpha 96 97  74 . . . 3229 3 (antigen CD49C, alpha 3subunit of VLA-3 receptor), variant a M454 ITGA3: integrin, alpha 98 99 74 . . . 3274 3 (antigen CD49C, alpha 3 subunit of VLA-3 receptor),variant b M35 ITGB6: integrin, 100 101  195 . . . 2561 beta 6 M36 KATII:L-kynurenine/ 102 103  454 . . . 1731 alpha-aminoadipateaminotransferase M666 KCNAB1: potassium 104 105  89 . . . 1315voltage-gated channel, shaker-related sub- family, beta member 1,variant 1 M667 KCNAB1: potassium 106 107  54 . . . 1313 voltage-gatedchannel, shaker-related sub- family, beta member 1, variant 2 M668KCNAB1: potassium 108 109  28 . . . 1233 voltage-gated channel,shaker-related sub- family, beta member 1, variant 3 M37 KIAA0662: 110111  <1 . . . 2035 KIAA0662 protein M38 LAMA3: Laminin, 112 113   1 . .. 5142 alpha-3 (nicein (150kD), (kalinin (165kD), BM600 (150kD) M39LAMC2: laminin, 114 115  90 . . . 3671 gamma 2 M40 LSM5: U6 snRNA- 116117   1 . . . 276 associated Sm-like protein M41 LUM: lumican 118 119 85 . . . 1101 M42 MACMARCKS: 120 121  14 . . . 601 macrophagemyristoyl- ated alanine-rich C kinase substrate M43 MAGP:microfibrillar- 122 123  115 . . . 666 associated protein 2 precursor,transcript variant 1 M44 MAGP: microfibrillar- 124 125  100 . . . 651associated protein 2 precursor, transcript variant 2 M45 MAPK: mitogen-126 127  328 . . . 1410 activated protein kinase 1 M489 MCM6:minichromo- 128 129  62 . . . 2527 some maintenance deficient (mis5, S.pombe) 6 M46 MDK: midkine 130 131  26 . . . 457 (neurite growth-promoting factor 2) M47 MGP: matrix Gla 132 133  47 . . . 358 proteinM48 MMP12: matrix 134 135  13 . . . 1425 metalloproteinase 12 M49 MMP3:matrix 136 137  64 . . . 1497 metalloproteinase 3, stromelysin 1,progel- atinase M294 MMP7: matrix 138 139  48 . . . 851metalloproteinase 7 (matrilysin, uterine), PUMP1 proteinase, variant 1OV52 MMP7: matrix 140 139  28 . . . 831 metalloproteinase 7 (matrilysin,uterine), PUMP1 proteinase, variant 2 M50 MMP9: matrix 141 142  20 . . .2143 metalloproteinase 9, gelatinase B, 92kD gelatinase, 92kD type IVcollagenase OV68 MSLN: mesothelin, 143 144  88 . . . 2196 variant 1 OV69MSLN: mesothelin, 145 146  88 . . . 1980 variant 2 OV70 MSLN:mesothelin, 147 148  88 . . . 1950 variant 3 OV71 MSLN: mesothelin, 149150  88 . . . 2172 variant 4 OV72 MSLN: mesothelin, 151 152  88 . . .1926 variant 5 OV43 MSLN: mesothelin, 153 154  88 . . . 1956 variant 6OV45 MUC1: mucin 1, 155 156  58 . . . 1605 transmembrane, variant 1 M669MUC1: mucin 1, 157 158  74 . . . 3841 transmembrane, variant 2 M51MYBL2: v-myb avian 159 160  128 . . . 2230 myeloblastosis viral oncogenehomolog-like 2 M52 MYH11: smooth 161 162  89 . . . 6007 muscle myosinheavy chain 11, isoform SM1 M53 MYH11: smooth 163 164  89 . . . 5905muscle myosin heavy chain 11, isoform SM2 M54 NK4: natural killer cell165 166  60 . . . 764 transcript 4, variant 1 M670 NK4: natural killercell 167 168  60 . . . 764 transcript 4 , variant 2 M55 NP25: (neuronal169 170  50 . . . 898 protein) OV48 OPN-a (osteopontin), 171 172   1 . .. 942 SPP1 (secreted phosphoprotein 1), bone sialoprotein I OV49 OPN-b(osteopontin), 173 174  88 . . . 990 SPP1 (secreted phosphoprotein 1),bone sialoprotein I OV50 OPN-c (osteopontin), 175 176   1 . . . 861 SPP1(secreted phosphoprotein 1), bone sialoprotein I M56 OSF-2, osteoblast177 178  12 . . . 2522 specific factor 2 (fasciclin I-like), variant 1M491 OSF-2, osteoblast 179 180  28 . . . 2367 specific factor 2(fasciclin I-like), variant 2 M57 PIM2: pim-2 oncogene 181 182  186 . .. 1190 M58 PLAU: plasminogen 183 184  77 . . . 1372 activator, urokinaseM59 PLK: polo (Droso- 185 186  64 . . . 1875 phia)-like kinase M671 PNN:pinin, desmo- 187 188  31 . . . 2262 some associated protein M60 PRG1:proteoglycan 1, 189 190  25 . . . 501 secretory granule M61 PTHLH:parathyroid 191 192  304 . . . 831 hormone-like hormone M62 PTN:pleiotrophin 193 194 1542 . . . 2048 (heparin binding growth factor 8,neurite growth- promoting factor 1) M63 RAB6KIFL: RAB6 195 196  28 . . .2700 interacting, kinesin- like (rabkinesin6) M64 RARRES3: retinoic 197198  62 . . . 556 acid receptor responder (tazarotene induced) 3 M65RBP1: retinol-binding 199 200  126 . . . 533 protein 1(cellular),CRABP-I, CRBP-I M66 RGS16: Regulator of 201 202  93 . . . 701 G proteinsignaling-16 M67 S100A2: S100 calcium 203 204  72 . . . 362 bindingprotein A2, variant 1 M68 S100A2: S100 calcium 205 206  41 . . . 334binding protein A2, variant 2 M69 SCYA20: small induc- 207 208  59 . . .349 ible cytokine sub- family A (Cys-Cys), member 20 M70 SPARC:Osteonectin 209 210  58 . . . 969 (secreted protein, acidic,cysteine-rich) M71 STCH: stress 70 211 212  37 . . . 1452 proteinchaperone, microsome-associated M492 STK12: serine/threo- 213 214  58 .. . 1092 nine kinase 12 M72 TK1: thymidine kinase 215 216  58 . . . 7621, soluble OV86 TMPRSS4: trans- 217 218  310 . . . 1623 membraneprotease, serine 4 M73 TMSB4X: thymosin, 219 220  78 . . . 212 beta 4, Xchromosome M74 TOP2A: topoiso- 221 222  37 . . . 4632 merase (DNA) IIalpha (170kD) M493 TPM1: tropomyosin 1 223 224  57 . . . 911 (alpha) M75TXN: thioredoxin 225 226  64 . . . 381 M76 UBCH10: ubiquitin 227 228  41. . . 580 carrier protein E2-C M77 UBD: diubiquitin 229 230  19 . . .516 M78 unnamed gene (1) 231 232  45 . . . 1353 M79 unnamed gene (2) 233234   1 . . . 1508 M80 VATD: vacuolar 235 236  166 . . . 909 proton pumpdelta polypeptide M81 ZWINT: ZW10 237 238  25 . . . 858 interactor

[0059] TABLE 2 SEQ ID SEQ ID Marker Gene Name NO (nts) NO (AAs) CDS M661AKAP9: A kinase 1 2 223 . . . 11946 (PRKA) anchor protein (yotiao) 9,variant 1 M662 AKAP9: A kinase 3 4 223 . . . 11922 (PRKA) anchor protein(yotiao) 9, variant 2 M663 AKAP9: A kinase 5 6 223 . . . 12000 (PRKA)anchor protein (yotiao) 9, variant 3 M664 AKAP9: A kinase 7 8 223 . . .11976 (PRKA) anchor protein (yotiao) 9, variant 4 OV68 MSLN: mesothelin,143 144  88 . . . 2196 variant 1 OV69 MSLN: mesothelin, 145 146  88 . .. 1980 variant 2 OV70 MSLN: mesothelin, 147 148  88 . . . 1950 variant 3OV71 MSLN: mesothelin, 149 150  88 . . . 2172 variant 4 OV72 MSLN:mesothelin, 151 152  88 . . . 1926 variant 5 M670 NK4: natural killercell 167 168  60 . . . 764 transcript 4 , variant 2 M67 S100A2: S100calcium 203 204  72 . . . 362 binding protein A2, variant 1 OV86TMPRSS4: trans- 217 218 310 . . . 1623 membrane protease, serine 4 M78unnamed gene (1) 231 232  45 . . . 1353 M79 unnamed gene (2) 233 234  1. . . 1508

[0060] TABLE 3 SEQ ID SEQ ID Marker Gene Name NO (nts) NO (AAs) CDS M481EPPK1: epiplakin 1 51 52  89 . . . 15286 M482 FOSL2: FOS-like 65 66 324. . . 1304 antigen 2, variant 1 M483 FOSL2: FOS-like 67 66 324 . . .1304 antigen 2, variant 2 M484 FSHPRH1: FSH 68 69 270 . . . 2540 primaryresponse (LRPR1, rat) homolog 1 M35 ITGB6: integrin, beta 100 101 195 .. . 2561 6 OV43 MSLN: mesothelin, 153 154  88 . . . 1956 variant 6

[0061] Definitions

[0062] As used herein, each of the following terms has the meaningassociated with it in this section.

[0063] The articles “a” and “an” are used herein to refer to one or tomore than one (i.e. to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

[0064] A “marker” is a gene whose altered level of expression in atissue or cell from its expression level in normal or healthy tissue orcell is associated with a disease state, such as cancer. A “markernucleic acid” is a nucleic acid (e.g., MRNA, cDNA) encoded by orcorresponding to a marker of the invention. Such marker nucleic acidsinclude DNA (e.g., cDNA) comprising the entire or a partial sequence ofany of the nucleic acid sequences set forth in the Sequence Listing orthe complement of such a sequence. The marker nucleic acids also includeRNA comprising the entire or a partial sequence of any of the nucleicacid sequences set forth in the Sequence Listing or the complement ofsuch a sequence, wherein all thymidine residues are replaced withuridine residues. A “marker protein” is a protein encoded by orcorresponding to a marker of the invention. A marker protein comprisesthe entire or a partial sequence of any of the sequences set forth inthe Sequence Listing. The terms “protein” and “polypeptide” are usedinterchangeably.

[0065] The term “probe” refers to any molecule which is capable ofselectively binding to a specifically intended target molecule, forexample, a nucleotide transcript or protein encoded by or correspondingto a marker. Probes can be either synthesized by one skilled in the art,or derived from appropriate biological preparations. For purposes ofdetection of the target molecule, probes may be specifically designed tobe labeled, as described herein. Examples of molecules that can beutilized as probes include, but are not limited to, RNA, DNA, proteins,antibodies, and organic molecules.

[0066] A “cervical-associated” body fluid is a fluid which, when in thebody of a patient, contacts or passes through cervical cells or intowhich cells or proteins shed from cervical cells are capable of passing.The cells may be found in a cervical smear collected, for example, by acervical brush. Exemplary cervical-associated body fluids include bloodfluids, lymph, ascitic fluids, gynecological fluids, cystic fluid,urine, and fluids collected by vaginal rinsing.

[0067] The “normal” level of expression of a marker is the level ofexpression of the marker in cervical cells of a human subject or patientnot afflicted with cervical cancer An “over-expression” or“significantly higher level of expression” of a marker refers to anexpression level in a test sample that is greater than the standarderror of the assay employed to assess expression, and is preferably atleast twice, and more preferably three, four, five or ten times theexpression level of the marker in a control sample (e.g., sample from ahealthy subjects not having the marker associated disease) andpreferably, the average expression level of the marker in severalcontrol samples.

[0068] A “significantly lower level of expression” of a marker refers toan expression level in a test sample that is at least twice, and morepreferably three, four, five or ten times lower than the expressionlevel of the marker in a control sample (e.g., sample from a healthysubject not having the marker associated disease) and preferably, theaverage expression level of the marker in several control samples.

[0069] As used herein, the term “promoter/regulatory sequence” means anucleic acid sequence which is required for expression of a gene productoperably linked to the promoter/regulatory sequence. In some instances,this sequence may be the core promoter sequence and in other instances,this sequence may also include an enhancer sequence and other regulatoryelements which are required for expression of the gene product. Thepromoter/regulatory sequence may, for example, be one which expressesthe gene product in a tissue-specific manner.

[0070] A “constitutive” promoter is a nucleotide sequence which, whenoperably linked with a polynucleotide which encodes or specifies a geneproduct, causes the gene product to be produced in a living human cellunder most or all physiological conditions of the cell.

[0071] An “inducible” promoter is a nucleotide sequence which, whenoperably linked with a polynucleotide which encodes or specifies a geneproduct, causes the gene product to be produced in a living human cellsubstantially only when an inducer which corresponds to the promoter ispresent in the cell.

[0072] A “tissue-specific” promoter is a nucleotide sequence which, whenoperably linked with a polynucleotide which encodes or specifies a geneproduct, causes the gene product to be produced in a living human cellsubstantially only if the cell is a cell of the tissue typecorresponding to the promoter.

[0073] A “transcribed polynucleotide” or “nucleotide transcript” is apolynucleotide (e.g. an MRNA, hnRNA, a cDNA, or an analog of such RNA orcDNA) which is complementary to or homologous with all or a portion of amature MRNA made by transcription of a marker of the invention andnormal post-transcriptional processing (e.g. splicing), if any, of theRNA transcript, and reverse transcription of the RNA transcript.

[0074] “Complementary” refers to the broad concept of sequencecomplementarity between regions of two nucleic acid strands or betweentwo regions of the same nucleic acid strand. It is known that an adenineresidue of a first nucleic acid region is capable of forming specifichydrogen bonds (“base pairing”) with a residue of a second nucleic acidregion which is antiparallel to the first region if the residue isthymine or uracil. Similarly, it is known that a cytosine residue of afirst nucleic acid strand is capable of base pairing with a residue of asecond nucleic acid strand which is antiparallel to the first strand ifthe residue is guanine. A first region of a nucleic acid iscomplementary to a second region of the same or a different nucleic acidif, when the two regions are arranged in an antiparallel fashion, atleast one nucleotide residue of the first region is capable of basepairing with a residue of the second region. Preferably, the firstregion comprises a first portion and the second region comprises asecond portion, whereby, when the first and second portions are arrangedin an antiparallel fashion, at least about 50%, and preferably at leastabout 75%, at least about 90%, or at least about 95% of the nucleotideresidues of the first portion are capable of base pairing withnucleotide residues in the second portion. More preferably, allnucleotide residues of the first portion are capable of base pairingwith nucleotide residues in the second portion.

[0075] “Homologous” as used herein, refers to nucleotide sequencesimilarity between two regions of the same nucleic acid strand orbetween regions of two different nucleic acid strands. When a nucleotideresidue position in both regions is occupied by the same nucleotideresidue, then the regions are homologous at that position. A firstregion is homologous to a second region if at least one nucleotideresidue position of each region is occupied by the same residue.Homology between two regions is expressed in terms of the proportion ofnucleotide residue positions of the two regions that are occupied by thesame nucleotide residue. By way of example, a region having thenucleotide sequence 5′-ATTGCC-3′ and a region having the nucleotidesequence 5′-TATGGC-3′ share 50% homology. Preferably, the first regioncomprises a first portion and the second region comprises a secondportion, whereby, at least about 50%, and preferably at least about 75%,at least about 90%, or at least about 95% of the nucleotide residuepositions of each of the portions are occupied by the same nucleotideresidue. More preferably, all nucleotide residue positions of each ofthe portions are occupied by the same nucleotide residue.

[0076] A molecule is “fixed” or “affixed” to a substrate if it iscovalently or non-covalently associated with the substrate such thesubstrate can be rinsed with a fluid (e.g. standard saline citrate, pH7.4) without a substantial fraction of the molecule dissociating fromthe substrate.

[0077] As used herein, a “naturally-occurring” nucleic acid moleculerefers to an RNA or DNA molecule having a nucleotide sequence thatoccurs in an organism found in nature.

[0078] A cancer is “inhibited” if at least one symptom of the cancer isalleviated, terminated, slowed, or prevented. As used herein, cervicalcancer is also “inhibited” if recurrence or metastasis of the cancer isreduced, slowed, delayed, or prevented.

[0079] A kit is any manufacture (e.g. a package or container) comprisingat least one reagent, e.g. a probe, for specifically detecting theexpression of a marker of the invention. The kit may be promoted,distributed, or sold as a unit for performing the methods of the presentinvention.

[0080] “Proteins of the invention” encompass marker proteins and theirfragments; variant marker proteins and their fragments; peptides andpolypeptides comprising an at least 15 amino acid segment of a marker orvariant marker protein; and fusion proteins comprising a marker orvariant marker protein, or an at least 15 amino acid segment of a markeror variant marker protein.

[0081] Unless otherwise specified here within, the terms “antibody” and“antibodies” broadly encompass naturally-occurring forms of antibodies(e.g. IgG, IgA, IgM, IgE) and recombinant antibodies such assingle-chain antibodies, chimeric and humanized antibodies andmulti-specific antibodies, as well as fragments and derivatives of allof the foregoing, which fragments and derivatives have at least anantigenic binding site. Antibody derivatives may comprise a protein orchemical moiety conjugated to an antibody.

[0082] Description

[0083] The present invention is based, in part, on newly identifiedmarkers which are over-expressed in cervical cancer cells as compared totheir expression in normal (i.e. non-cancerous) cervical cells. Theenhanced expression of one or more of these markers in cervical cells isherein correlated with the cancerous state of the tissue. The inventionprovides compositions, kits, and methods for assessing the cancerousstate of cervical cells (e.g. cells obtained from a human, culturedhuman cells, archived or preserved human cells and in vivo cells) aswell as treating patients afflicted with cervical cancer.

[0084] The compositions, kits, and methods of the invention have thefollowing uses, among others:

[0085] 1) assessing whether a patient is afflicted with cervical cancer;

[0086] 2) assessing the stage of cervical cancer in a human patient;

[0087] 3) assessing the grade of cervical cancer in a patient;

[0088] 4) assessing the benign or malignant nature of cervical cancer ina patient;

[0089] 5) assessing the metastatic potential of cervical cancer in apatient;

[0090] 6) assessing the histological type of neoplasm associated withcervical cancer in a patient;

[0091] 7) making antibodies, antibody fragments or antibody derivativesthat are useful for treating cervical cancer and/or assessing whether apatient is afflicted with cervical cancer;

[0092] 8) assessing the presence of cervical cancer cells;

[0093] 9) assessing the efficacy of one or more test compounds forinhibiting cervical cancer in a patient;

[0094] 10) assessing the efficacy of a therapy for inhibiting cervicalcancer in a patient;

[0095] 11) monitoring the progression of cervical cancer in a patient;

[0096] 12) selecting a composition or therapy for inhibiting cervicalcancer in a patient;

[0097] 13) treating a patient afflicted with cervical cancer;

[0098] 14) inhibiting cervical cancer in a patient;

[0099] 15) assessing the cervical carcinogenic potential of a testcompound; and

[0100] 16) preventing the onset of cervical cancer in a patient at riskfor developing cervical cancer.

[0101] The invention thus includes a method of assessing whether apatient is afflicted with cervical cancer which includes assessingwhether the patient has pre-metastasized cervical cancer. This methodcomprises comparing the level of expression of a marker of the invention(listed in Table 1) in a patient sample and the normal level ofexpression of the marker in a control, e.g., a non-cervical cancersample. A significantly higher level of expression of the marker in thepatient sample as compared to the normal level is an indication that thepatient is afflicted with cervical cancer.

[0102] Gene delivery vehicles, host cells and compositions (alldescribed herein) containing nucleic acids comprising the entirety, or asegment of 15 or more nucleotides, of any of the nucleic acid sequencesset forth in the Sequence Listing, or the complement of such sequences,and polypeptides comprising the entirety, or a segment of 10 or moreamino acids, of any of the amino acid sequences set forth in theSequence Listing, are also provided by this invention.

[0103] As described herein, cervical cancer in patients is associatedwith an increased level of expression of one or more markers of theinvention. While, as discussed above, some of these changes inexpression level result from occurrence of the cervical cancer, othersof these changes induce, maintain, and promote the cancerous state ofcervical cancer cells. Thus, cervical cancer characterized by anincrease in the level of expression of one or more markers of theinvention can be inhibited by reducing and/or interfering with theexpression of the markers and/or function of the proteins encoded bythose markers.

[0104] Expression of a marker of the invention can be inhibited in anumber of ways generally known in the art. For example, an antisenseoligonucleotide can be provided to the cervical cancer cells in order toinhibit transcription, translation, or both, of the marker(s).Alternately, a polynucleotide encoding an antibody, an antibodyderivative, or an antibody fragment which specifically binds a markerprotein, and operably linked with an appropriate promoter/regulatorregion, can be provided to the cell in order to generate intracellularantibodies which will inhibit the function or activity of the protein.The expression and/or function of a marker may also be inhibited bytreating the cervical cancer cell with an antibody, antibody derivativeor antibody fragment that specifically binds a marker protein. Using themethods described herein, a variety of molecules, particularly includingmolecules sufficiently small that they are able to cross the cellmembrane, can be screened in order to identify molecules which inhibitexpression of a marker or inhibit the function of a marker protein. Thecompound so identified can be provided to the patient in order toinhibit cervical cancer cells of the patient.

[0105] Any marker or combination of markers of the invention, as well asany known markers in combination with the markers of the invention, maybe used in the compositions, kits, and methods of the present invention.In general, it is preferable to use markers for which the differencebetween the level of expression of the marker in cervical cancer cellsand the level of expression of the same marker in normal cervical cellsis as great as possible. Although this difference can be as small as thelimit of detection of the method for assessing expression of the marker,it is preferred that the difference be at least greater than thestandard error of the assessment method, and preferably a difference ofat least 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 15-, 20-, 100-, 500-,1000-fold or greater than the level of expression of the same marker innormal cervical tissue.

[0106] It is recognized that certain marker proteins are secreted fromcervical cells (i.e. one or both of normal and cancerous cells) to theextracellular space surrounding the cells. These markers are preferablyused in certain embodiments of the compositions, kits, and methods ofthe invention, owing to the fact that the such marker proteins can bedetected in a cervical-associated body fluid sample, which may be moreeasily collected from a human patient than a tissue biopsy sample. Inaddition, preferred in vivo techniques for detection of a marker proteininclude introducing into a subject a labeled antibody directed againstthe protein. For example, the antibody can be labeled with a radioactivemarker whose presence and location in a subject can be detected bystandard imaging techniques.

[0107] It is a simple matter for the skilled artisan to determinewhether any particular marker protein is a secreted protein. In order tomake this determination, the marker protein is expressed in, forexample, a mammalian cell, preferably a human cervical cell line,extracellular fluid is collected, and the presence or absence of theprotein in the extracellular fluid is assessed (e.g. using a labeledantibody which binds specifically with the protein).

[0108] The following is an example of a method which can be used todetect secretion of a protein. About 8×10 293T cells are incubated at37° C. in wells containing growth medium (Dulbecco's modified Eagle'smedium {DMEM} supplemented with 10% fetal bovine serum) under a 5% (v/v)CO₂, 95% air atmosphere to about 60-70% confluence. The cells are thentransfected using a standard transfection mixture comprising 2micrograms of DNA comprising an expression vector encoding the proteinand 10 microliters of LipofectAMINETm (GIBCO/BRL Catalog no. 18342-012)per well. The transfection mixture is maintained for about 5 hours, andthen replaced with fresh growth medium and maintained in an airatmosphere. Each well is gently rinsed twice with DMEM which does notcontain methionine or cysteine (DMEM-MC; ICN Catalog no. 16-424-54).About 1 milliliter of DMEM-MC and about 50 microcuries of Trans-³⁵S™reagent (ICN Catalog no. 51006) are added to each well. The wells aremaintained under the 5% CO₂ atmosphere described above and incubated at37° C. for a selected period. Following incubation, 150 microliters ofconditioned medium is removed and centrifuged to remove floating cellsand debris. The presence of the protein in the supernatant is anindication that the protein is secreted.

[0109] It will be appreciated that patient samples containing cervicalcells may be used in the methods of the present invention. In theseembodiments, the level of expression of the marker can be assessed byassessing the amount (e.g. absolute amount or concentration) of themarker in a cervical cell sample, e.g., cervical smear obtained from apatient. The cell sample can, of course, be subjected to a variety ofwell-known post-collection preparative and storage techniques (e.g.,nucleic acid and/or protein extraction, fixation, storage, freezing,ultrafiltration, concentration, evaporation, centrifugation, etc.) priorto assessing the amount of the marker in the sample. Likewise, cervicalsmears may also be subjected to post-collection preparative and storagetechniques, e.g., fixation.

[0110] The compositions, kits, and methods of the invention can be usedto detect expression of marker proteins having at least one portionwhich is displayed on the surface of cells which express it. It is asimple matter for the skilled artisan to determine whether a markerprotein, or a portion thereof, is exposed on the cell surface. Forexample, immunological methods may be used to detect such proteins onwhole cells, or well known computer-based sequence analysis methods maybe used to predict the presence of at least one extracellular domain(i.e. including both secreted proteins and proteins having at least onecell-surface domain). Expression of a marker protein having at least oneportion which is displayed on the surface of a cell which expresses itmay be detected without necessarily lysing the cell (e.g. using alabeled antibody which binds specifically with a cell-surface domain ofthe protein).

[0111] Expression of a marker of the invention may be assessed by any ofa wide variety of well known methods for detecting expression of atranscribed nucleic acid or protein. Non-limiting examples of suchmethods include immunological methods for detection of secreted,cell-surface, cytoplasmic, or nuclear proteins, protein purificationmethods, protein finction or activity assays, nucleic acid hybridizationmethods, nucleic acid reverse transcription methods, and nucleic acidamplification methods.

[0112] In a preferred embodiment, expression of a marker is assessedusing an antibody (e.g. a radio-labeled, chromophore-labeled,fluorophore-labeled, or enzyme-labeled antibody), an antibody derivative(e.g. an antibody conjugated with a substrate or with the protein orligand of a protein-ligand pair {e.g. biotin-streptavidin}), or anantibody fragment (e.g. a single-chain antibody, an isolated antibodyhypervariable domain, etc.) which binds specifically with a markerprotein or fragment thereof, including a marker protein which hasundergone all or a portion of its normal post-translationalmodification.

[0113] In another preferred embodiment, expression of a marker isassessed by preparing mRNA/cDNA (i.e. a transcribed polynucleotide) fromcells in a patient sample, and by hybridizing the mRNA/cDNA with areference polynucleotide which is a complement of a marker nucleic acid,or a fragment thereof. cDNA can, optionally, be amplified using any of avariety of polymerase chain reaction methods prior to hybridization withthe reference polynucleotide; preferably, it is not amplified.Expression of one or more markers can likewise be detected usingquantitative PCR to assess the level of expression of the marker(s).Alternatively, any of the many known methods of detecting mutations orvariants (e.g. single nucleotide polymorphisms, deletions, etc.) of amarker of the invention may be used to detect occurrence of a marker ina patient.

[0114] In a related embodiment, a mixture of transcribed polynucleotidesobtained from the sample is contacted with a substrate having fixedthereto a polynucleotide complementary to or homologous with at least aportion (e.g. at least 7, 10, 15, 20, 25, 30, 40, 50, 100, 500, or morenucleotide residues) of a marker nucleic acid. If polynucleotidescomplementary to or homologous with are differentially detectable on thesubstrate (e.g. detectable using different chromophores or fluorophores,or fixed to different selected positions), then the levels of expressionof a plurality of markers can be assessed simultaneously using a singlesubstrate (e.g. a “gene chip” microarray of polynucleotides fixed atselected positions). When a method of assessing marker expression isused which involves hybridization of one nucleic acid with another, itis preferred that the hybridization be performed under stringenthybridization conditions.

[0115] Because the compositions, kits, and methods of the invention relyon detection of a difference in expression levels of one or more markersof the invention, it is preferable that the level of expression of themarker is significantly greater than the minimum detection limit of themethod used to assess expression in at least one of normal cervicalcells and cancerous cervical cells.

[0116] It is understood that by routine screening of additional patientsamples using one or more of the markers of the invention, it will berealized that certain of the markers are over-expressed in cancers ofvarious types, including specific cervical cancers, as well as othercancers such as breast cancer, ovarian cancer, etc. For example, it willbe confirmed that some of the markers of the invention areover-expressed in most (i.e. 50% or more) or substantially all (i.e. 80%or more) of cervical cancer. Furthermore, it will be confirmed thatcertain of the markers of the invention are associated with cervicalcancer of various stages (i.e. stage 0, I, II, III, and IV cervicalcancers, as well as subclassifications IAl, IA2, IB, IB1, IB2, IIA, IIB,IIIA, IIIB, IVA, and IVB, using the FIGO Stage Grouping system forprimary carcinoma of the cervix (see Gynecologic Oncology, 1991, 41:199and Cancer, 1992, 69:482)), and pre-malignant conditions (e.g.,dysplasia including CIN or SIL), of various histologic subtypes (e.g.squamous cell carcinomas and squamous cell carcinoma variants such asverrucous carcinoma, lymphoepithelioma-like carcinoma, papillarysquamous neoplasm and spindle cell squamous cell carcinoma (see CervicalCancer and Preinvasive Neoplasia, 1996, pp. 90-91) serous, mucinous,endometrioid, and clear cell subtypes, as well as subclassifications andalternate classifications adenocarcinoma, papillary adenocarcinoma,papillary cystadenocarcinoma, surface papillary carcinoma, malignantadenofibroma, cystadenofibroma, adenocarcinoma, cystadenocarcinoma,adenoacanthoma, endometrioid stromal sarcoma, mesodermal {Müllerian}mixed tumor, malignant carcinoma, Brenner tumor, mixed epithelial tumor,and undifferentiated carcinoma, using the WHO/FIGO system forclassification of malignant cervical tumors; Scully, Atlas of TumorPathology, 3d series, Washington D.C.), and various grades (i.e. grade I{well differentiated} , grade II {moderately well differentiated}, andgrade III {poorly differentiated from surrounding normal tissue}). Inaddition, as a greater number of patient samples are assessed forexpression of the markers of the invention and the outcomes of theindividual patients from whom the samples were obtained are correlated,it will also be confirmed that altered expression of certain of themarkers of the invention are strongly correlated with malignant cancersand that altered expression of other markers of the invention arestrongly correlated with benign tumors. The compositions, kits, andmethods of the invention are thus useful for characterizing one or moreof the stage, grade, histological type, and benign/malignant nature ofcervical cancer in patients.

[0117] When the compositions, kits, and methods of the invention areused for characterizing one or more of the stage, grade, histologicaltype, and benign/malignant nature of cervical cancer in a patient, it ispreferred that the marker or panel of markers of the invention isselected such that a positive result is obtained in at least about 20%,and preferably at least about 40%, 60%, or 80%, and more preferably insubstantially all patients afflicted with a cervical cancer of thecorresponding stage, grade, histological type, or benign/malignantnature. Preferably, the marker or panel of markers of the invention isselected such that a positive predictive value (PPV) of greater thanabout 10% is obtained for the general population (more preferablycoupled with an assay specificity greater than 80%).

[0118] When a plurality of markers of the invention are used in thecompositions, kits, and methods of the invention, the level ofexpression of each marker in a patient sample can be compared with thenormal level of expression of each of the plurality of markers innon-cancerous samples of the same type, either in a single reactionmixture (i.e. using reagents, such as different fluorescent probes, foreach marker) or in individual reaction mixtures corresponding to one ormore of the markers. In one embodiment, a significantly increased levelof expression of more than one of the plurality of markers in thesample, relative to the corresponding normal levels, is an indicationthat the patient is afflicted with cervical cancer. When a plurality ofmarkers is used, it is preferred that 2, 3, 4, 5, 8, 10, 12, 15, 20, 30,or 50 or more individual markers be used, wherein fewer markers arepreferred.

[0119] In order to maximize the sensitivity of the compositions, kits,and methods of the invention (i.e. by interference attributable to cellsof non-cervical origin in a patient sample), it is preferable that themarker of the invention used therein be a marker which has a restrictedtissue distribution, e.g., normally not expressed in a non-cervicaltissue.

[0120] Only a small number of markers are known to be associated withcervical cancer (e.g. bcl-2, 15A8 antigen, cdc6, Mcm5, and EGFR). Thesemarkers are not, of course, included among the markers of the invention,although they may be used together with one or more markers of theinvention in a panel of markers, for example. It is well known thatcertain types of genes, such as oncogenes, tumor suppressor genes,growth factor-like genes, protease-like genes, and protein kinase-likegenes are often involved with development of cancers of various types.Thus, among the markers of the invention, use of those which correspondto proteins which resemble known proteins encoded by known oncogenes andtumor suppressor genes, and those which correspond to proteins whichresemble growth factors, proteases, and protein kinases are preferred.

[0121] It is recognized that the compositions, kits, and methods of theinvention will be of particular utility to patients having an enhancedrisk of developing cervical cancer and their medical advisors. Patientsrecognized as having an enhanced risk of developing cervical cancerinclude, for example, patients having a familial history of cervicalcancer, patients identified as having a mutant oncogene (i.e. at leastone allele), and patients of advancing age (i.e. women older than about50 or 60 years).

[0122] The level of expression of a marker in normal (i.e.non-cancerous) human cervical tissue can be assessed in a variety ofways. In one embodiment, this normal level of expression is assessed byassessing the level of expression of the marker in a portion of cervicalcells which appears to be non-cancerous and by comparing this normallevel of expression with the level of expression in a portion of thecervical cells which is suspected of being cancerous. Alternately, andparticularly as further information becomes available as a result ofroutine performance of the methods described herein, population-averagevalues for normal expression of the markers of the invention may beused. In other embodiments, the ‘normal’ level of expression of a markermay be determined by assessing expression of the marker in a patientsample obtained from a non-cancer-afflicted patient, from a patientsample obtained from a patient before the suspected onset of cervicalcancer in the patient, from archived patient samples, and the like.

[0123] The invention includes compositions, kits, and methods forassessing the presence of cervical cancer cells in a sample (e.g. anarchived tissue sample or a sample obtained from a patient). Thesecompositions, kits, and methods are substantially the same as thosedescribed above, except that, where necessary, the compositions, kits,and methods are adapted for use with samples other than patient samples.For example, when the sample to be used is a parafinized, archived humantissue sample, it can be necessary to adjust the ratio of compounds inthe compositions of the invention, in the kits of the invention, or themethods used to assess levels of marker expression in the sample. Suchmethods are well known in the art and within the skill of the ordinaryartisan.

[0124] The invention includes a kit for assessing the presence ofcervical cancer cells (e.g. in a sample such as a patient sample). Thekit comprises a plurality of reagents, each of which is capable ofbinding specifically with a marker nucleic acid or protein. Suitablereagents for binding with a marker protein include antibodies, antibodyderivatives, antibody fragments, and the like. Suitable reagents forbinding with a marker nucleic acid (e.g. a genomic DNA, an MRNA, aspliced MRNA, a cDNA, or the like) include complementary nucleic acids.For example, the nucleic acid reagents may include oligonucleotides(labeled or non-labeled) fixed to a substrate, labeled oligonucleotidesnot bound with a substrate, pairs of PCR primers, molecular beaconprobes, and the like.

[0125] The kit of the invention may optionally comprise additionalcomponents useful for performing the methods of the invention. By way ofexample, the kit may comprise fluids (e.g. SSC buffer) suitable forannealing complementary nucleic acids or for binding an antibody with aprotein with which it specifically binds, one or more samplecompartments, an instructional material which describes performance of amethod of the invention, a sample of normal cervical cells, a sample ofcervical cancer cells, and the like.

[0126] The invention also includes a method of making an isolatedhybridoma which produces an antibody useful for assessing whetherpatient is afflicted with an cervical cancer. In this method, a proteinor peptide comprising the entirety or a segment of a marker protein issynthesized or isolated (e.g. by purification from a cell in which it isexpressed or by transcription and translation of a nucleic acid encodingthe protein or peptide in vivo or in vitro using known methods). Avertebrate, preferably a mammal such as a mouse, rat, rabbit, or sheep,is immunized using the protein or peptide. The vertebrate may optionally(and preferably) be immunized at least one additional time with theprotein or peptide, so that the vertebrate exhibits a robust immuneresponse to the protein or peptide. Splenocytes are isolated from theimmunized vertebrate and fused with an immortalized cell line to formhybridomas, using any of a variety of methods well known in the art.Hybridomas formed in this manner are then screened using standardmethods to identify one or more hybridomas which produce an antibodywhich specifically binds with the marker protein or a fragment thereof.The invention also includes hybridomas made by this method andantibodies made using such hybridomas.

[0127] The invention also includes a method of assessing the efficacy ofa test compound for inhibiting cervical cancer cells. As describedabove, differences in the level of expression of the markers of theinvention correlate with the cancerous state of cervical cells. Althoughit is recognized that changes in the levels of expression of certain ofthe markers of the invention likely result from the cancerous state ofcervical cells, it is likewise recognized that changes in the levels ofexpression of other of the markers of the invention induce, maintain,and promote the cancerous state of those cells. Thus, compounds whichinhibit an cervical cancer in a patient will cause the level ofexpression of one or more of the markers of the invention to change to alevel nearer the normal level of expression for that marker (i.e. thelevel of expression for the marker in non-cancerous cervical cells).

[0128] This method thus comprises comparing expression of a marker in afirst cervical cell sample and maintained in the presence of the testcompound and expression of the marker in a second cervical cell sampleand maintained in the absence of the test compound. A significantlyreduced expression of a marker of the invention in the presence of thetest compound is an indication that the test compound inhibits cervicalcancer. The cervical cell samples may, for example, be aliquots of asingle sample of normal cervical cells obtained from a patient, pooledsamples of normal cervical cells obtained from a patient, cells of anormal cervical cell line, aliquots of a single sample of cervicalcancer cells obtained from a patient, pooled samples of cervical cancercells obtained from a patient, cells of an cervical cancer cell line, orthe like. In one embodiment, the samples are cervical cancer cellsobtained from a patient and a plurality of compounds known to beeffective for inhibiting various cervical cancers are tested in order toidentify the compound which is likely to best inhibit the cervicalcancer in the patient.

[0129] This method may likewise be used to assess the efficacy of atherapy for inhibiting cervical cancer in a patient. In this method, thelevel of expression of one or more markers of the invention in a pair ofsamples (one subjected to the therapy, the other not subjected to thetherapy) is assessed. As with the method of assessing the efficacy oftest compounds, if the therapy induces a significantly lower level ofexpression of a marker of the invention then the therapy is efficaciousfor inhibiting cervical cancer. As above, if samples from a selectedpatient are used in this method, then alternative therapies can beassessed in vitro in order to select a therapy most likely to beefficacious for inhibiting cervical cancer in the patient.

[0130] As described above, the cancerous state of human cervical cellsis correlated with changes in the levels of expression of the markers ofthe invention. The invention includes a method for assessing the humancervical cell carcinogenic potential of a test compound. This methodcomprises maintaining separate aliquots of human cervical cells in thepresence and absence of the test compound. Expression of a marker of theinvention in each of the aliquots is compared. A significantly higherlevel of expression of a marker of the invention in the aliquotmaintained in the presence of the test compound (relative to the aliquotmaintained in the absence of the test compound) is an indication thatthe test compound possesses human cervical cell carcinogenic potential.The relative carcinogenic potentials of various test compounds can beassessed by comparing the degree of enhancement or inhibition of thelevel of expression of the relevant markers, by comparing the number ofmarkers for which the level of expression is enhanced or inhibited, orby comparing both.

[0131] Various aspects of the invention are described in further detailin the following subsections.

[0132] I. Isolated Nucleic Acid Molecules

[0133] One aspect of the invention pertains to isolated nucleic acidmolecules, including nucleic acids which encode a marker protein or aportion thereof. Isolated nucleic acids of the invention also includenucleic acid molecules sufficient for use as hybridization probes toidentify marker nucleic acid molecules, and fragments of marker nucleicacid molecules, e.g., those suitable for use as PCR primers for theamplification or mutation of marker nucleic acid molecules. As usedherein, the term “nucleic acid molecule” is intended to include DNAmolecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., MRNA) andanalogs of the DNA or RNA generated using nucleotide analogs. Thenucleic acid molecule can be single-stranded or double-stranded, butpreferably is double-stranded DNA.

[0134] An “isolated” nucleic acid molecule is one which is separatedfrom other nucleic acid molecules which are present in the naturalsource of the nucleic acid molecule. Preferably, an “isolated” nucleicacid molecule is free of sequences (preferably protein-encodingsequences) which naturally flank the nucleic acid (i.e., sequenceslocated at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA ofthe organism from which the nucleic acid is derived. For example, invarious embodiments, the isolated nucleic acid molecule can contain lessthan about 5 kB, 4 kB, 3 kB, 2 kB, 1 kB, 0.5 kB or 0.1 kB of nucleotidesequences which naturally flank the nucleic acid molecule in genomic DNAof the cell from which the nucleic acid is derived. Moreover, an“isolated” nucleic acid molecule, such as a CDNA molecule, can besubstantially free of other cellular material, or culture medium whenproduced by recombinant techniques, or substantially free of chemicalprecursors or other chemicals when chemically synthesized.

[0135] A nucleic acid molecule of the present invention can be isolatedusing standard molecular biology techniques and the sequence informationin the database records described herein. Using all or a portion of suchnucleic acid sequences, nucleic acid molecules of the invention can beisolated using standard hybridization and cloning techniques (e.g., asdescribed in Sambrook et al., ed., Molecular Cloning: A LaboratoryManual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989).

[0136] A nucleic acid molecule of the invention can be amplified usingcDNA, MRNA, or genomic DNA as a template and appropriate oligonucleotideprimers according to standard,PCR amplification techniques. The nucleicacid so amplified can be cloned into an appropriate vector andcharacterized by DNA sequence analysis. Furthermore, nucleotidescorresponding to all or a portion of a nucleic acid molecule of theinvention can be prepared by standard synthetic techniques, e.g., usingan automated DNA synthesizer.

[0137] In another preferred embodiment, an isolated nucleic acidmolecule of the invention comprises a nucleic acid molecule which has anucleotide sequence complementary to the nucleotide sequence of a markernucleic acid or to the nucleotide sequence of a nucleic acid encoding amarker protein. A nucleic acid molecule which is complementary to agiven nucleotide sequence is one which is sufficiently complementary tothe given nucleotide sequence that it can hybridize to the givennucleotide sequence thereby forming a stable duplex.

[0138] Moreover, a nucleic acid molecule of the invention can compriseonly a portion of a nucleic acid sequence, wherein the full lengthnucleic acid sequence comprises a marker nucleic acid or which encodes amarker protein. Such nucleic acids can be used, for example, as a probeor primer. The probe/primer typically is used as one or moresubstantially purified oligonucleotides. The oligonucleotide typicallycomprises a region of nucleotide sequence that hybridizes understringent conditions to at least about 7, preferably about 15, morepreferably about 25, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, or400 or more consecutive nucleotides of a nucleic acid of the invention.

[0139] Probes based on the sequence of a nucleic acid molecule of theinvention can be used to detect transcripts or genomic sequencescorresponding to one or more markers of the invention. The probecomprises a label group attached thereto, e.g., a radioisotope, afluorescent compound, an enzyme, or an enzyme co-factor. Such probes canbe used as part of a diagnostic test kit for identifying cells ortissues which misexpress the protein, such as by measuring levels of anucleic acid molecule encoding the protein in a sample of cells from asubject, e.g., detecting MRNA levels or determining whether a geneencoding the protein has been mutated or deleted.

[0140] The invention further encompasses nucleic acid molecules thatdiffer, due to degeneracy of the genetic code, from the nucleotidesequence of nucleic acids encoding a marker protein (e.g., a proteinhaving one of the amino acid sequences set forth in the SequenceListing), and thus encode the same protein.

[0141] It will be appreciated by those skilled in the art that DNAsequence polymorphisms that lead to changes in the amino acid sequencecan exist within a population (e.g., the human population). Such geneticpolymorphisms can exist among individuals within a population due tonatural allelic variation. An allele is one of a group of genes whichoccur alternatively at a given genetic locus. In addition, it will beappreciated that DNA polymorphisms that affect RNA expression levels canalso exist that may affect the overall expression level of that gene(e.g., by affecting regulation or degradation).

[0142] As used herein, the phrase “allelic variant” refers to anucleotide sequence which occurs at a given locus or to a polypeptideencoded by the nucleotide sequence.

[0143] As used herein, the terms “gene” and “recombinant gene” refer tonucleic acid molecules comprising an open reading frame encoding apolypeptide corresponding to a marker of the invention. Such naturalallelic variations can typically result in 1-5% variance in thenucleotide sequence of a given gene. Alternative alleles can beidentified by sequencing the gene of interest in a number of differentindividuals. This can be readily carried out by using hybridizationprobes to identify the same genetic locus in a variety of individuals.Any and all such nucleotide variations and resulting amino acidpolymorphisms or variations that are the result of natural allelicvariation and that do not alter the functional activity are intended tobe within the scope of the invention.

[0144] In another embodiment, an isolated nucleic acid molecule of theinvention is at least 7, 15, 20, 25, 30, 40, 60, 80, 100, 150, 200, 250,300, 350, 400, 450, 550, 650, 700, 800, 900, 1000, 1200, 1400, 1600,1800, 2000, 2200, 2400, 2600, 2800, 3000, 3500, 4000, 4500, or morenucleotides in length and hybridizes under stringent conditions to amarker nucleic acid or to a nucleic acid encoding a marker protein. Asused herein, the term “hybridizes under stringent conditions” isintended to describe conditions for hybridization and washing underwhich nucleotide sequences at least 60% (65%, 70%, preferably 75%)identical to each other typically remain hybridized to each other. Suchstringent conditions are known to those skilled in the art and can befound in sections 6.3.1-6.3.6 of Current Protocols in Molecular Biology,John Wiley & Sons, N.Y. (1989). A preferred, non-limiting example ofstringent hybridization conditions are hybridization in 6×sodiumchloride/sodium citrate (SSC) at about 45° C., followed by one or morewashes in 0.2×SSC, 0.1% SDS at 50-65° C.

[0145] In addition to naturally-occurring allelic variants of a nucleicacid molecule of the invention that can exist in the population, theskilled artisan will further appreciate that sequence changes can beintroduced by mutation thereby leading to changes in the amino acidsequence of the encoded protein, without altering the biologicalactivity of the protein encoded thereby. For example, one can makenucleotide substitutions leading to amino acid substitutions at“non-essential” amino acid residues. A “non-essential” amino acidresidue is a residue that can be altered from the wild-type sequencewithout altering the biological activity, whereas an “essential” aminoacid residue is required for biological activity. For example, aminoacid residues that are not conserved or only semi-conserved amonghomologs of various species may be non-essential for activity and thuswould be likely targets for alteration. Alternatively, amino acidresidues that are conserved among the homologs of various species (e.g.,murine and human) may be essential for activity and thus would not belikely targets for alteration.

[0146] Accordingly, another aspect of the invention pertains to nucleicacid molecules encoding a variant marker protein that contain changes inamino acid residues that are not essential for activity. Such variantmarker proteins differ in amino acid sequence from thenaturally-occurring marker proteins, yet retain biological activity. Inone embodiment, such a variant marker protein has an amino acid sequencethat is at least about 40% identical, 50%, 60%, 70%, 80%, 90%, 95%, or98% identical to the amino acid sequence of a marker protein.

[0147] An isolated nucleic acid molecule encoding a variant markerprotein can be created by introducing one or more nucleotidesubstitutions, additions or deletions into the nucleotide sequence ofmarker nucleic acids, such that one or more amino acid residuesubstitutions, additions, or deletions are introduced into the encodedprotein. Mutations can be introduced by standard techniques, such assite-directed mutagenesis and PCR-mediated mutagenesis. Preferably,conservative amino acid substitutions are made at one or more predictednon-essential amino acid residues. A “conservative amino acidsubstitution” is one in which the amino acid residue is replaced with anamino acid residue having a similar side chain. Families of amino acidresidues having similar side chains have been defined in the art. Thesefamilies include amino acids with basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Alternatively, mutations can beintroduced randomly along all or part of the coding sequence, such as bysaturation mutagenesis, and the resultant mutants can be screened forbiological activity to identify mutants that retain activity. Followingmutagenesis, the encoded protein can be expressed recombinantly and theactivity of the protein can be determined.

[0148] The present invention encompasses antisense nucleic acidmolecules, i.e., molecules which are complementary to a sense nucleicacid of the invention, e.g., complementary to the coding strand of adouble-stranded marker cDNA molecule or complementary to a marker mRNAsequence. Accordingly, an antisense nucleic acid of the invention canhydrogen bond to (i.e. anneal with) a sense nucleic acid of theinvention. The antisense nucleic acid can be complementary to an entirecoding strand, or to only a portion thereof, e.g., all or part of theprotein coding region (or open reading frame). An antisense nucleic acidmolecule can also be antisense to all or part of a non-coding region ofthe coding strand of a nucleotide sequence encoding a marker protein.The non-coding regions (“5′ and 3′ untranslated regions”) are the 5′ and3′ sequences which flank the coding region and are not translated intoamino acids.

[0149] An antisense oligonucleotide can be, for example, about 5, 10,15, 20, 25, 30, 35, 40, 45, or 50 or more nucleotides in length. Anantisense nucleic acid of the invention can be constructed usingchemical synthesis and enzymatic ligation reactions using proceduresknown in the art. For example, an antisense nucleic acid (e.g., anantisense oligonucleotide) can be chemically synthesized using naturallyoccurring nucleotides or variously modified nucleotides designed toincrease the biological stability of the molecules or to increase thephysical stability of the duplex formed between the antisense and sensenucleic acids, e.g., phosphorothioate derivatives and acridinesubstituted nucleotides can be used. Examples of modified nucleotideswhich can be used to generate the antisense nucleic acid include5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxyhnethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been sub-cloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following subsection).

[0150] The antisense nucleic acid molecules of the invention aretypically administered to a subject or generated in situ such that theyhybridize with or bind to cellular mRNA and/or genomic DNA encoding amarker protein to thereby inhibit expression of the marker, e.g., byinhibiting transcription and/or translation. The hybridization can be byconventional nucleotide complementarity to form a stable duplex, or, forexample, in the case of an antisense nucleic acid molecule which bindsto DNA duplexes, through specific interactions in the major groove ofthe double helix. Examples of a route of administration of antisensenucleic acid molecules of the invention includes direct injection at atissue site or infusion of the antisense nucleic acid into anovary-associated body fluid. Alternatively, antisense nucleic acidmolecules can be modified to target selected cells and then administeredsystemically. For example, for systemic administration, antisensemolecules can be modified such that they specifically bind to receptorsor antigens expressed on a selected cell surface, e.g., by linking theantisense nucleic acid molecules to peptides or antibodies which bind tocell surface receptors or antigens. The antisense nucleic acid moleculescan also be delivered to cells using the vectors described herein. Toachieve sufficient intracellular concentrations of the antisensemolecules, vector constructs in which the antisense nucleic acidmolecule is placed under the control of a strong pol II or pol IIIpromoter are preferred.

[0151] An antisense nucleic acid molecule of the invention can be anα-anomeric nucleic acid molecule. An α-anomeric nucleic acid moleculeforms specific double-stranded hybrids with complementary RNA in which,contrary to the usual α-units, the strands run parallel to each other(Gaultier et al., 1987, Nucleic Acids Res. 15:6625-6641). The antisensenucleic acid molecule can also comprise a 2′-o-methylribonucleotide(Inoue et al., 1987, Nucleic Acids Res. 15:6131-6148) or a chimericRNA-DNA analogue (Inoue et aL, 1987, FEBS Lett. 215:327-330).

[0152] The invention also encompasses ribozymes. Ribozymes are catalyticRNA molecules with ribonuclease activity which are capable of cleaving asingle-stranded nucleic acid, such as an mRNA, to which they have acomplementary region. Thus, ribozymes (e.g., hammerhead ribozymes asdescribed in Haselhoff and Gerlach, 1988, Nature 334:585-591) can beused to catalytically cleave MRNA transcripts to thereby inhibittranslation of the protein encoded by the mRNA. A ribozyme havingspecificity for a nucleic acid molecule encoding a marker protein can bedesigned based upon the nucleotide sequence of a cDNA corresponding tothe marker. For example, a derivative of a Tetrahymena L-19 IVS RNA canbe constructed in which the nucleotide sequence of the active site iscomplementary to the nucleotide sequence to be cleaved (see Cech et aLU.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742).Alternatively, an mRNA encoding a polypeptide of the invention can beused to select a catalytic RNA having a specific ribonuclease activityfrom a pool of RNA molecules (see, e.g., Bartel and Szostak, 1993,Science 261:1411-1418).

[0153] The invention also encompasses nucleic acid molecules which formtriple helical structures. For example, expression of a marker of theinvention can be inhibited by targeting nucleotide sequencescomplementary to the regulatory region of the gene encoding the markernucleic acid or protein (e.g., the promoter and/or enhancer) to formtriple helical structures that prevent transcription of the gene intarget cells. See generally Helene (1991) Anticancer Drug Des.6(6):569-84; Helene (1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher(1992) Bioassays 14(12):807-15.

[0154] In various embodiments, the nucleic acid molecules of theinvention can be modified at the base moiety, sugar moiety or phosphatebackbone to improve, e.g., the stability, hybridization, or solubilityof the molecule. For example, the deoxyribose phosphate backbone of thenucleic acids can be modified to generate peptide nucleic acids (seeHyrup et al., 1996, Bioorganic & Medicinal Chemistry 4(1): 5-23). Asused herein, the terms “peptide nucleic acids” or “PNAs” refer tonucleic acid mimics, e.g., DNA mimics, in which the deoxyribosephosphate backbone is replaced by a pseudopeptide backbone and only thefour natural nucleobases are retained. The neutral backbone of PNAs hasbeen shown to allow for specific hybridization to DNA and RNA underconditions of low ionic strength. The synthesis of PNA oligomers can beperformed using standard solid phase peptide synthesis protocols asdescribed in Hyrup et al. (1996), supra; Perry-O'Keefe et aL (1996)Proc. Natl. Acad. Sci. USA 93:14670-675.

[0155] PNAs can be used in therapeutic and diagnostic applications. Forexample, PNAs can be used as antisense or antigene agents forsequence-specific modulation of gene expression by, e.g., inducingtranscription or translation arrest or inhibiting replication. PNAs canalso be used, e.g., in the analysis of single base pair mutations in agene by, e.g., PNA directed PCR clamping; as artificial restrictionenzymes when used in combination with other enzymes, e.g., S1 nucleases(Hyrup (1996), supra; or as probes or primers for DNA sequence andhybridization (Hyrup, 1996, supra; Perry-O'Keefe et al., 1996, Proc.Natl. Acad. Sci. USA 93:14670-675).

[0156] In another embodiment, PNAs can be modified, e.g., to enhancetheir stability or cellular uptake, by attaching lipophilic or otherhelper groups to PNA, by the formation of PNA-DNA chimeras, or by theuse of liposomes or other techniques of drug delivery known in the art.For example, PNA-DNA chimeras can be generated which can combine theadvantageous properties of PNA and DNA. Such chimeras allow DNArecognition enzymes, e.g., RNase H and DNA polymerases, to interact withthe DNA portion while the PNA portion would provide high bindingaffinity and specificity. PNA-DNA chimeras can be linked using linkersof appropriate lengths selected in terms of base stacking, number ofbonds between the nucleobases, and orientation (Hyrup, 1996, supra). Thesynthesis of PNA-DNA chimeras can be performed as described in Hyrup(1996), supra, and Finn et al. (1996) Nucleic Acids Res. 24(17):3357-63.For example, a DNA chain can be synthesized on a solid support usingstandard phosphoramidite coupling chemistry and modified nucleosideanalogs. Compounds such as 5′-(4-methoxytrityl)amino-5′-deoxy-thymidinephosphoramidite can be used as a link between the PNA and the 5′ end ofDNA (Mag et al., 1989, Nucleic Acids Res. 17:5973-88). PNA monomers arethen coupled in a step-wise manner to produce a chimeric molecule with a5′ PNA segment and a 3′ DNA segment (Finn et al., 1996, Nucleic AcidsRes. 24(17):3357-63). Alternatively, chimeric molecules can besynthesized with a 5′ DNA segment and a 3′ PNA segment (Peterser et al.,1975, Bioorganic Med. Chem. Lett. 5:1119-11124).

[0157] In other embodiments, the oligonucleotide can include otherappended groups such as peptides (e.g., for targeting host cellreceptors in vivo), or agents facilitating transport across the cellmembrane (see, e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci. USA86:6553-6556; Lemaitre et al., 1987, Proc. Natl. Acad. Sci. USA84:648-652; PCT Publication No. WO 88/09810) or the blood-brain barrier(see, e.g., PCT Publication No. WO 89/10134). In addition,oligonucleotides can be modified with hybridization-triggered cleavageagents (see, e.g., Krol et al., 1988, Bio/Techniques 6:958-976) orintercalating agents (see, e.g., Zon, 1988, Pharm. Res. 5:539-549). Tothis end, the oligonucleotide can be conjugated to another molecule,e.g., a peptide, hybridization triggered cross-linking agent, transportagent, hybridization-triggered cleavage agent, etc.

[0158] The invention also includes molecular beacon nucleic acids havingat least one region which is complementary to a nucleic acid of theinvention, such that the molecular beacon is useful for quantitating thepresence of the nucleic acid of the invention in a sample. A “molecularbeacon” nucleic acid is a nucleic acid comprising a pair ofcomplementary regions and having a fluorophore and a fluorescentquencher associated therewith. The fluorophore and quencher areassociated with different portions of the nucleic acid in such anorientation that when the complementary regions are annealed with oneanother, fluorescence of the fluorophore is quenched by the quencher.When the complementary regions of the nucleic acid are not annealed withone another, fluorescence of the fluorophore is quenched to a lesserdegree. Molecular beacon nucleic acids are described, for example, inU.S. Pat. No. 5,876,930.

[0159] II. Isolated Proteins and Antibodies

[0160] One aspect of the invention pertains to isolated marker proteinsand biologically active portions thereof, as well as polypeptidefragments suitable for use as immunogens to raise antibodies directedagainst a marker protein or a fragment thereof. In one embodiment, thenative marker protein can be isolated from cells or tissue sources by anappropriate purification scheme using standard protein purificationtechniques. In another embodiment, a protein or peptide comprising thewhole or a segment of the marker protein is produced by recombinant DNAtechniques. Alternative to recombinant expression, such protein orpeptide can be synthesized chemically using standard peptide synthesistechniques.

[0161] An “isolated” or “purified” protein or biologically activeportion thereof is substantially free of cellular material or othercontaminating proteins from the cell or tissue source from which theprotein is derived, or substantially free of chemical precursors orother chemicals when chemically synthesized. The language “substantiallyfree of cellular material” includes preparations of protein in which theprotein is separated from cellular components of the cells from which itis isolated or recombinantly produced. Thus, protein that issubstantially free of cellular material includes preparations of proteinhaving less than about 30%, 20%, 10%, or 5% (by dry weight) ofheterologous protein (also referred to herein as a “contaminatingprotein”). When the protein or biologically active portion thereof isrecombinantly produced, it is also preferably substantially free ofculture medium, i.e., culture medium represents less than about 20%,10%, or 5% of the volume of the protein preparation. When the protein isproduced by chemical synthesis, it is preferably substantially free ofchemical precursors or other chemicals, i.e., it is separated fromchemical precursors or other chemicals which are involved in thesynthesis of the protein. Accordingly such preparations of the proteinhave less than about 30%, 20%, 10%, 5% (by dry weight) of chemicalprecursors or compounds other than the polypeptide of interest.

[0162] Biologically active portions of a marker protein includepolypeptides comprising amino acid sequences sufficiently identical toor derived from the amino acid sequence of the marker protein, whichinclude fewer amino acids than the full length protein, and exhibit atleast one activity of the corresponding full-length protein. Typically,biologically active portions comprise a domain or motif with at leastone activity of the corresponding full-length protein. A biologicallyactive portion of a marker protein of the invention can be a polypeptidewhich is, for example, 10, 25, 50, 100 or more amino acids in length.Moreover, other biologically active portions, in which other regions ofthe marker protein are deleted, can be prepared by recombinanttechniques and evaluated for one or more of the functional activities ofthe native form of the marker protein.

[0163] Preferred marker proteins are encoded by nucleotide sequencescomprising the sequence of any of the sequences set forth in theSequence Listing. Other useful proteins are substantially identical(e.g., at least about 40%, preferably 50%, 60%, 70%, 80%, 90%, 95%, or99%) to one of these sequences and retain the finctional activity of thecorresponding naturally-occurring marker protein yet differ in aminoacid sequence due to natural allelic variation or mutagenesis.

[0164] To determine the percent identity of two amino acid sequences orof two nucleic acids, the sequences are aligned for optimal comparisonpurposes (e.g. gaps can be introduced in the sequence of a first aminoacid or nucleic acid sequence for optimal alignment with a second aminoor nucleic acid sequence). The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences (i.e., % identity=# ofidentical positions/total # of positions (e.g., overlappingpositions)×100). In one embodiment the two sequences are the samelength.

[0165] The determination of percent identity between two sequences canbe accomplished using a mathematical algorithm. A preferred,non-limiting example of a mathematical algorithm utilized for thecomparison of two sequences is the algorithm of Karlin and Altschul(1990) Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlinand Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Such analgorithm is incorporated into the BLASTN and BLASTX programs ofAltschul, et al. (1990) J. Mol. Biol. 215:403-410. BLAST nucleotidesearches can be performed with the BLASTN program, score=100,wordlength=12 to obtain nucleotide sequences homologous to a nucleicacid molecules of the invention. BLAST protein searches can be performedwith the BLASTP program, score=50, wordlength=3 to obtain amino acidsequences homologous to a protein molecules of the invention. To obtaingapped alignments for comparison purposes, a newer version of the BLASTalgorithm called Gapped BLAST can be utilized as described in Altschulet al. (1997) Nucleic Acids Res. 25:3389-3402, which is able to performgapped local alignments for the programs BLASTN, BLASTP and BLASTX.Alternatively, PSI-Blast can be used to perform an iterated search whichdetects distant relationships between molecules. When utilizing BLAST,Gapped BLAST, and PSI-Blast programs, the default parameters of therespective programs (e.g., BLASTX and BLASTN) can be used. See http://www.ncbi.nlm.nih.gov. Another preferred, non-limiting example of amathematical algorithm utilized for the comparison of sequences is thealgorithm of Myers and Miller, (1988) CABIOS 4:11-17. Such an algorithmis incorporated into the ALIGN program (version 2.0) which is part ofthe GCG sequence alignment software package. When utilizing the ALIGNprogram for comparing amino acid sequences, a PAM120 weight residuetable, a gap length penalty of 12, and a gap penalty of 4 can be used.Yet another useful algorithm for identifying regions of local sequencesimilarity and alignment is the FASTA algorithm as described in Pearsonand Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444-2448. When usingthe FASTA algorithm for comparing nucleotide or amino acid sequences, aPAM120 weight residue table can, for example, be used with a k-tuplevalue of 2.

[0166] The percent identity between two sequences can be determinedusing techniques similar to those described above, with or withoutallowing gaps. In calculating percent identity, only exact matches arecounted.

[0167] The invention also provides chimeric or fusion proteinscomprising a marker protein or a segment thereof As used herein, a“chimeric protein” or “fusion protein” comprises all or part (preferablya biologically active part) of a marker protein operably linked to aheterologous polypeptide (i.e., a polypeptide other than the markerprotein). Within the fusion protein, the term “operably linked” isintended to indicate that the marker protein or segment thereof and theheterologous polypeptide are fused in-frame to each other. Theheterologous polypeptide can be fused to the aminoterminus or thecarboxyl-terminus of the marker protein or segment.

[0168] One useful fusion protein is a GST fusion protein in which amarker protein or segment is fused to the carboxyl terminus of GSTsequences. Such fusion proteins can facilitate the purification of arecombinant polypeptide of the invention.

[0169] In another embodiment, the fusion protein contains a heterologoussignal sequence at its amino terminus. For example, the native signalsequence of a marker protein can be removed and replaced with a signalsequence from another protein. For example, the gp67 secretory sequenceof the baculovirus envelope protein can be used as a heterologous signalsequence (Ausubel et al., ed., Current Protocols in Molecular Biology,John Wiley & Sons, N.Y., 1992). Other examples of eukaryoticheterologous signal sequences include the secretory sequences ofmelittin and human placental alkaline phosphatase (Stratagene; La Jolla,Calif.). In yet another example, useful prokaryotic heterologous signalsequences include the phoA secretory signal (Sambrook et aL., supra) andthe protein A secretory signal (Pharmacia Biotech; Piscataway, N.J.).

[0170] In yet another embodiment, the fusion protein is animmunoglobulin fusion protein in which all or part of a marker proteinis fused to sequences derived from a member of the immunoglobulinprotein family. The immunoglobulin fusion proteins of the invention canbe incorporated into pharmaceutical compositions and administered to asubject to inhibit an interaction between a ligand (soluble ormembrane-bound) and a protein on the surface of a cell (receptor), tothereby suppress signal transduction in vivo. The immunoglobulin fusionprotein can be used to affect the bioavailability of a cognate ligand ofa marker protein. Inhibition of ligand/receptor interaction can beuseful therapeutically, both for treating proliferative anddifferentiative disorders and for modulating (e.g. promoting orinhibiting) cell survival. Moreover, the immunoglobulin fusion proteinsof the invention can be used as immunogens to produce antibodiesdirected against a marker protein in a subject, to purify ligands and inscreening assays to identify molecules which inhibit the interaction ofthe marker protein with ligands.

[0171] Chimeric and fusion proteins of the invention can be produced bystandard recombinant DNA techniques. In another embodiment, the fusiongene can be synthesized by conventional techniques including automatedDNA synthesizers. Alternatively, PCR amplification of gene fragments canbe carried out using anchor primers which give rise to complementaryoverhangs between two consecutive gene fragments which can subsequentlybe annealed and re-amplified to generate a chimeric gene sequence (see,e.g., Ausubel et al., supra). Moreover, many expression vectors arecommercially available that already encode a fusion moiety (e.g., a GSTpolypeptide). A nucleic acid encoding a polypeptide of the invention canbe cloned into such an expression vector such that the fusion moiety islinked in-frame to the polypeptide of the invention.

[0172] A signal sequence can be used to facilitate secretion andisolation of marker proteins. Signal sequences are typicallycharacterized by a core of hydrophobic amino acids which are generallycleaved from the mature protein during secretion in one or more cleavageevents. Such signal peptides contain processing sites that allowcleavage of the signal sequence from the mature proteins as they passthrough the secretory pathway. Thus, the invention pertains to markerproteins, fusion proteins or segments thereof having a signal sequence,as well as to such proteins from which the signal sequence has beenproteolytically cleaved (i.e., the cleavage products). In oneembodiment, a nucleic acid sequence encoding a signal sequence can beoperably linked in an expression vector to a protein of interest, suchas a marker protein or a segment thereof. The signal sequence directssecretion of the protein, such as from a eukaryotic host into which theexpression vector is transformed, and the signal sequence issubsequently or concurrently cleaved. The protein can then be readilypurified from the extracellular medium by art recognized methods.Alternatively, the signal sequence can be linked to the protein ofinterest using a sequence which facilitates purification, such as with aGST domain.

[0173] The present invention also pertains to variants of the markerproteins. Such variants have an altered amino acid sequence which canfunction as either agonists (mimetics) or as antagonists. Variants canbe generated by mutagenesis, e.g. discrete point mutation or truncation.An agonist can retain substantially the same, or a subset, of thebiological activities of the naturally occurring form of the protein. Anantagonist of a protein can inhibit one or more of the activities of thenaturally occurring form of the protein by, for example, competitivelybinding to a downstream or upstream member of a cellular signalingcascade which includes the protein of interest. Thus, specificbiological effects can be elicited by treatment with a variant oflimited function. Treatment of a subject with a variant having a subsetof the biological activities of the naturally occurring form of theprotein can have fewer side effects in a subject relative to treatmentwith the naturally occurring form of the protein.

[0174] Variants of a marker protein which function as either agonists(mimetics) or as antagonists can be identified by screeningcombinatorial libraries of mutants, e.g., truncation mutants, of theprotein of the invention for agonist or antagonist activity. In oneembodiment, a variegated library of variants is generated bycombinatorial mutagenesis at the nucleic acid level and is encoded by avariegated gene library. A variegated library of variants can beproduced by, for example, enzymatically ligating a mixture of syntheticoligonucleotides into gene sequences such that a degenerate set ofpotential protein sequences is expressible as individual polypeptides,or alternatively, as a set of larger fusion proteins (e.g., for phagedisplay). There are a variety of methods which can be used to producelibraries of potential variants of the marker proteins from a degenerateoligonucleotide sequence. Methods for synthesizing degenerateoligonucleotides are known in the art (see, e.g., Narang, 1983,Tetrahedron 39:3; Itakura et al., 1984, Annu. Rev. Biochem. 53:323;Itakura et al., 1984, Science 198:1056; Ike et al., 1983 Nucleic AcidRes. 11:477).

[0175] In addition, libraries of segments of a marker protein can beused to generate a variegated population of polypeptides for screeningand subsequent selection of variant marker proteins or segments thereof.For example, a library of coding sequence fragments can be generated bytreating a double stranded PCR fragment of the coding sequence ofinterest with a nuclease under conditions wherein nicking occurs onlyabout once per molecule, denaturing the double stranded DNA, renaturingthe DNA to form double stranded DNA which can include sense/antisensepairs from different nicked products, removing single stranded portionsfrom reformed duplexes by treatment with S1 nuclease, and ligating theresulting fragment library into an expression vector. By this method, anexpression library can be derived which encodes amino terminal andinternal fragments of various sizes of the protein of interest.

[0176] Several techniques are known in the art for screening geneproducts of combinatorial libraries made by point mutations ortruncation, and for screening cDNA libraries for gene products having aselected property. The most widely used techniques, which are amenableto high through-put analysis, for screening large gene librariestypically include cloning the gene library into replicable expressionvectors, transforming appropriate cells with the resulting library ofvectors, and expressing the combinatorial genes under conditions inwhich detection of a desired activity facilitates isolation of thevector encoding the gene whose product was detected. Recursive ensemblemutagenesis (REM), a technique which enhances the frequency offunctional mutants in the libraries, can be used in combination with thescreening assays to identify variants of a protein of the invention(Arkin and Yourvan, 1992, Proc. Natl. Acad. Sci. USA 89:7811-7815;Delgrave et al., 1993, Protein Engineering 6(3):327-331).

[0177] Another aspect of the invention pertains to antibodies directedagainst a protein of the invention. In preferred embodiments, theantibodies specifically bind a marker protein or a fragment thereof. Theterms “antibody” and “antibodies” as used interchangeably herein referto immunoglobulin molecules as well as fragments and derivatives thereofthat comprise an immunologically active portion of an immunoglobulinmolecule, (i.e., such a portion contains an antigen binding site whichspecifically binds an antigen, such as a marker protein, e.g., anepitope of a marker protein). An antibody which specifically binds to aprotein of the invention is an antibody which binds the protein, butdoes not substantially bind other molecules in a sample, e.g., abiological sample, which naturally contains the protein. Examples of animmunologically active portion of an immunoglobulin molecule include,but are not limited to, single-chain antibodies (scAb), F(ab) andF(ab′)₂ fragments.

[0178] An isolated protein of the invention or a fragment thereof can beused as an immunogen to generate antibodies. The full-length protein canbe used or, alternatively, the invention provides antigenic peptidefragments for use as immunogens. The antigenic peptide of a protein ofthe invention comprises at least 8 (preferably 10, 15, 20, or 30 ormore) amino acid residues of the amino acid sequence of one of theproteins of the invention, and encompasses at least one epitope of theprotein such that an antibody raised against the peptide forms aspecific immune complex with the protein. Preferred epitopes encompassedby the antigenic peptide are regions that are located on the surface ofthe protein, e.g., hydrophilic regions. Hydrophobicity sequenceanalysis, hydrophilicity sequence analysis, or similar analyses can beused to identify hydrophilic regions. In preferred embodiments, anisolated marker protein or fragment thereof is used as an immunogen.

[0179] An immunogen typically is used to prepare antibodies byimmunizing a suitable (i.e. immunocompetent) subject such as a rabbit,goat, mouse, or other mammal or vertebrate. An appropriate immunogenicpreparation can contain, for example, recombinantly-expressed orchemically-synthesized protein or peptide. The preparation can furtherinclude an adjuvant, such as Freund's complete or incomplete adjuvant,or a similar immunostimulatory agent. Preferred immunogen compositionsare those that contain no other human proteins such as, for example,immunogen compositions made using a non-human host cell for recombinantexpression of a protein of the invention. In such a manner, theresulting antibody compositions have reduced or no binding of humanproteins other than a protein of the invention.

[0180] The invention provides polyclonal and monoclonal antibodies. Theterm “monoclonal antibody” or “monoclonal antibody composition”, as usedherein, refers to a population of antibody molecules that contain onlyone species of an antigen binding site capable of immunoreacting with aparticular epitope. Preferred polyclonal and monoclonal antibodycompositions are ones that have been selected for antibodies directedagainst a protein of the invention. Particularly preferred polyclonaland monoclonal antibody preparations are ones that contain onlyantibodies directed against a marker protein or fragment thereof.

[0181] Polyclonal antibodies can be prepared by immunizing a suitablesubject with a protein of the invention as an immunogen The antibodytiter in the immunized subject can be monitored over time by standardtechniques, such as with an enzyme linked immunosorbent assay (ELISA)using immobilized polypeptide. At an appropriate time afterimmunization, e.g., when the specific antibody titers are highest,antibody-producing cells can be obtained from the subject and used toprepare monoclonal antibodies (mAb) by standard techniques, such as thehybridoma technique originally described by Kohler and Milstein (1975)Nature 256:495-497, the human B cell hybridoma technique (see Kozbor etal., 1983, Immunol. Today 4:72), the EBV-hybridoma technique (see Coleet al., pp. 77-96 In Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, Inc., 1985) or trioma techniques. The technology for producinghybridomas is well known (see generally Current Protocols in Immunology,Coligan et al. ed., John Wiley & Sons, N.Y., 1994). Hybridoma cellsproducing a monoclonal antibody of the invention are detected byscreening the hybridoma culture supernatants for antibodies that bindthe polypeptide of interest, e.g., using a standard ELISA assay.

[0182] Alternative to preparing monoclonal antibody-secretinghybridomas, a monoclonal antibody directed against a protein of theinvention can be identified and isolated by screening a recombinantcombinatorial immunoglobulin library (e.g., an antibody phage displaylibrary) with the polypeptide of interest. Kits for generating andscreening phage display libraries are commercially available (e.g., thePharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; andthe Stratagene SurfZAP Phage Display Kit, Catalog No. 240612).Additionally, examples of methods and reagents particularly amenable foruse in generating and screening antibody display library can be foundin, for example, U.S. Pat. No. 5,223,409; PCT Publication No. WO92/18619; PCT Publication No. WO 91/17271; PCT Publication No. WO92/20791; PCT Publication No. WO 92/15679; PCT Publication No. WO93/01288; PCT Publication No. WO 92/01047; PCT Publication No. WO92/09690; PCT Publication No. WO 90/02809; Fuchs et al. (1991)Bio/Technology 9:1370-1372; Hay et al. (1992) Hum. Antibod. Hybridomas3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffiths et al.(1993) EMBO J. 12:725-734.

[0183] The invention also provides recombinant antibodies thatspecifically bind a protein of the invention. In preferred embodiments,the recombinant antibodies specifically binds a marker protein orfragment thereof. Recombinant antibodies include, but are not limitedto, chimeric and humanized monoclonal antibodies, comprising both humanand non-human portions, single-chain antibodies and multi-specificantibodies. A chimeric antibody is a molecule in which differentportions are derived from different animal species, such as those havinga variable region derived from a murine mAb and a human immunoglobulinconstant region. (See, e.g., Cabilly et al., U.S. Pat. No. 4,816,567;and Boss et al., U.S. Pat. No. 4,816,397, which are incorporated hereinby reference in their entirety.) Single-chain antibodies have an antigenbinding site and consist of a single polypeptide. They can be producedby techniques known in the art, for example using methods described inLadner et. al U.S. Pat. No. 4,946,778 (which is incorporated herein byreference in its entirety); Bird et al., (1988) Science 242:423-426;Whitlow et al., (1991) Methods in Enzymology 2:1-9; Whitlow et al.,(1991) Methods in Enzymology 2:97-105; and Huston et al., (1991) Methodsin Enzymology Molecular Design and Modeling: Concepts and Applications203:46-88. Multi-specific antibodies are antibody molecules having atleast two antigen-binding sites that specifically bind differentantigens. Such molecules can be produced by techniques known in the art,for example using methods described in Segal, U.S. Pat. No. 4,676,980(the disclosure of which is incorporated herein by reference in itsentirety); Holliger et al., (1993) Proc. Natl. Acad. Sci. USA90:6444-6448; Whitlow et al., (1994) Protein Eng. 7:1017-1026 and U.S.Pat. No. 6,121,424.

[0184] Humanized antibodies are antibody molecules from non-humanspecies having one or more complementarity determining regions (CDRs)from the non-human species and a framework region from a humanimmunoglobulin molecule. (See, e.g., Queen, U.S. Pat. No. 5,585,089,which is incorporated herein by reference in its entirety.) Humanizedmonoclonal antibodies can be produced by recombinant DNA techniquesknown in the art, for example using methods described in PCT PublicationNo. WO 87/02671; European Patent Application 184,187; European PatentApplication 171,496; European Patent Application 173,494; PCTPublication No. WO 86/01533; U.S. Pat. No. 4,816,567; European PatentApplication 125,023; Better et al. (1988) Science 240:1041-1043; Liu etal. (1987) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J.Immunol. 139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA84:214-218; Nishimura etaL. (1987) Cancer Res. 47:999-1005; Wood et al.(1985) Nature 314:446-449; and Shaw et al. (1988) J. Natl. Cancer Inst.80:1553-1559); Morrison (1985) Science 229:1202-1207; Oi et al. (1986)Bio/Techniques 4:214; U.S. Pat. No. 5,225,539; Jones et al. (1986)Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; andBeidler et al. (1988) J. Immunol. 141:4053-4060.

[0185] More particularly, humanized antibodies can be produced, forexample, using transgenic mice which are incapable of expressingendogenous immunoglobulin heavy and light chains genes, but which canexpress human heavy and light chain genes. The transgenic mice areimmunized in the normal fashion with a selected antigen, e.g., all or aportion of a polypeptide corresponding to a marker of the invention.Monoclonal antibodies directed against the antigen can be obtained usingconventional hybridoma technology. The human immunoglobulin transgenesharbored by the transgenic mice rearrange during B cell differentiation,and subsequently undergo class switching and somatic mutation. Thus,using such a technique, it is possible to produce therapeutically usefulIgG, IgA and IgE antibodies. For an overview of this technology forproducing human antibodies, see Lonberg and Huszar (1995) Int. Rev.Immunol. 13:65-93). For a detailed discussion of this technology forproducing human antibodies and human monoclonal antibodies and protocolsfor producing such antibodies, see, e.g., U.S. Pat. No. 5,625,126; U.S.Pat. No. 5,633,425; U.S. Pat. No. 5,569,825; U.S. Pat. No. 5,661,016;and U.S. Pat. No. 5,545,806. In addition, companies such as Abgenix,Inc. (Freemont, Calif.), can be engaged to provide human antibodiesdirected against a selected antigen using technology similar to thatdescribed above.

[0186] Completely human antibodies which recognize a selected epitopecan be generated using a technique referred to as “guided selection.” Inthis approach a selected non-human monoclonal antibody, e.g., a murineantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope (Jespers et al., 1994, Bio/technology12:899-903).

[0187] The antibodies of the invention can be isolated after production(e.g., from the blood or serum of the subject) or synthesis and furtherpurified by well-known techniques. For example, IgG antibodies can bepurified using protein A chromatography. Antibodies specific for aprotein of the invention can be selected or (e.g., partially purified)or purified by, e.g., affinity chromatography. For example, arecombinantly expressed and purified (or partially purified) protein ofthe invention is produced as described herein, and covalently ornon-covalently coupled to a solid support such as, for example, achromatography column. The column can then be used to affinity purifyantibodies specific for the proteins of the invention from a samplecontaining antibodies directed against a large number of differentepitopes, thereby generating a substantially purified antibodycomposition, i.e., one that is substantially free of contaminatingantibodies. By a substantially purified antibody composition is meant,in this context, that the antibody sample contains at most only 30% (bydry weight) of contaminating antibodies directed against epitopes otherthan those of the desired protein of the invention, and preferably atmost 20%, yet more preferably at most 10%, and most preferably at most5% (by dry weight) of the sample is contaminating antibodies. A purifiedantibody composition means that at least 99% of the antibodies in thecomposition are directed against the desired protein of the invention.

[0188] In a preferred embodiment, the substantially purified antibodiesof the invention may specifically bind to a signal peptide, a secretedsequence, an extracellular domain, a transmembrane or a cytoplasmicdomain or cytoplasmic membrane of a protein of the invention. In aparticularly preferred embodiment, the substantially purified antibodiesof the invention specifically bind to a secreted sequence or anextracellular domain of the amino acid sequences of a protein of theinvention. In a more preferred embodiment, the substantially purifiedantibodies of the invention specifically bind to a secreted sequence oran extracellular domain of the amino acid sequences of a marker protein.

[0189] An antibody directed against a protein of the invention can beused to isolate the protein by standard techniques, such as affinitychromatography or immunoprecipitation. Moreover, such an antibody can beused to detect the marker protein or fragment thereof (e.g., in acellular lysate or cell supernatant) in order to evaluate the level andpattern of expression of the marker. The antibodies can also be useddiagnostically to monitor protein levels in tissues or body fluids (e.g.in a cervical-associated body fluid) as part of a clinical testingprocedure, e.g., to, for example, determine the efficacy of a giventreatment regimen. Detection can be facilitated by the use of anantibody derivative, which comprises an antibody of the inventioncoupled to a detectable substance. Examples of detectable substancesinclude various enzymes, prosthetic groups, fluorescent materials,luminescent materials, bioluminescent materials, and radioactivematerials. Examples of suitable enzymes include horseradish peroxidase,alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examplesof suitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin,and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or³H.

[0190] Antibodies of the invention may also be used as therapeuticagents in treating cancers. In a preferred embodiment, completely humanantibodies of the invention are used for therapeutic treatment of humancancer patients, particularly those having an cervical cancer. Inanother preferred embodiment, antibodies that bind specifically to amarker protein or fragment thereof are used for therapeutic treatment.Further, such therapeutic antibody may be an antibody derivative orimmunotoxin comprising an antibody conjugated to a therapeutic moietysuch as a cytotoxin, a therapeutic agent or a radioactive metal ion. Acytotoxin or cytotoxic agent includes any agent that is detrimental tocells. Examples include taxol, cytochalasin B, gramicidin D, ethidiumbromide, emetine, mitomycin, etoposide, tenoposide, vincristine,vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracindione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone,glucocorticoids, procaine, tetracaine, lidocaine, propranolol, andpuromycin and analogs or homologs thereof. Therapeutic agents include,but are not limited to, antimetabolites (e.g., methotrexate,6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracildecarbazine), alkylating agents (e.g., mechlorethamine, thioepachlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cis-dichlorodiamine platinum (II) (DDP) cisplatin),anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

[0191] The conjugated antibodies of the invention can be used formodifying a given biological response, for the drug moiety is not to beconstrued as limited to classical chemical therapeutic agents. Forexample, the drug moiety may be a protein or polypeptide possessing adesired biological activity. Such proteins may include, for example, atoxin such as ribosome-inhibiting protein (see Better et al., U.S. Pat.No. 6,146,631, the disclosure of which is incorporated herein in itsentirety), abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; aprotein such as tumor necrosis factor, .alpha.-interferon,.beta.-interferon, nerve growth factor, platelet derived growth factor,tissue plasminogen activator; or, biological response modifiers such as,for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2(“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophase colonystimulating factor (“GM-CSF”), granulocyte colony stimulating factor(“G-CSF”), or other growth factors.

[0192] Techniques for conjugating such therapeutic moiety to antibodiesare well known, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies 84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev., 62:119-58 (1982).

[0193] Accordingly, in one aspect, the invention provides substantiallypurified antibodies, antibody fragments and derivatives, all of whichspecifically bind to a protein of the invention and preferably, a markerprotein. In various embodiments, the substantially purified antibodiesof the invention, or fragments or derivatives thereof, can be human,non-human, chimeric and/or humanized antibodies. In another aspect, theinvention provides non-human antibodies, antibody fragments andderivatives, all of which specifically bind to a protein of theinvention and preferably, a marker protein. Such non-human antibodiescan be goat, mouse, sheep, horse, chicken, rabbit, or rat antibodies.Alternatively, the non-human antibodies of the invention can be chimericand/or humanized antibodies. In addition, the non-human antibodies ofthe invention can be polyclonal antibodies or monoclonal antibodies. Instill a further aspect, the invention provides monoclonal antibodies,antibody fragments and derivatives, all of which specifically bind to aprotein of the invention and preferably, a marker protein. Themonoclonal antibodies can be human, humanized, chimeric and/or non-humanantibodies.

[0194] The invention also provides a kit containing an antibody of theinvention conjugated to a detectable substance, and instructions foruse. Still another aspect of the invention is a pharmaceuticalcomposition comprising an antibody of the invention. In one embodiment,the pharmaceutical composition comprises an antibody of the inventionand a pharmaceutically acceptable carrier.

[0195] III. Recombinant Expression Vectors and Host Cells

[0196] Another aspect of the invention pertains to vectors, preferablyexpression vectors, containing a nucleic acid encoding a marker protein(or a portion of such a protein). As used herein, the term “vector”refers to a nucleic acid molecule capable of transporting anothernucleic acid to which it has been linked. One type of vector is a“plasmid”, which refers to a circular double stranded DNA loop intowhich additional DNA segments can be ligated. Another type of vector isa viral vector, wherein additional DNA segments can be ligated into theviral genome. Certain vectors are capable of autonomous replication in ahost cell into which they are introduced (e.g., bacterial vectors havinga bacterial origin of replication and episomal mammalian vectors). Othervectors (e.g., non-episomal mammalian vectors) are integrated into thegenome of a host cell upon introduction into the host cell, and therebyare replicated along with the host genome. Moreover, certain vectors,namely expression vectors, are capable of directing the expression ofgenes to which they are operably linked. In general, expression vectorsof utility in recombinant DNA techniques are often in the form ofplasmids (vectors). However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

[0197] The recombinant expression vectors of the invention comprise anucleic acid of the invention in a form suitable for expression of thenucleic acid in a host cell. This means that the recombinant expressionvectors include one or more regulatory sequences, selected on the basisof the host cells to be used for expression, which is operably linked tothe nucleic acid sequence to be expressed. Within a recombinantexpression vector, “operably linked” is intended to mean that thenucleotide sequence of interest is linked to the regulatory sequence(s)in a manner which allows for expression of the nucleotide sequence(e.g., in an in vitro transcription/translation system or in a host cellwhen the vector is introduced into the host cell). The term “regulatorysequence” is intended to include promoters, enhancers and otherexpression control elements (e.g., polyadenylation signals). Suchregulatory sequences are described, for example, in Goeddel, Methods inEnzymology: Gene Expression Technology vol.185, Academic Press, SanDiego, Calif. (1991). Regulatory sequences include those which directconstitutive expression of a nucleotide sequence in many types of hostcell and those which direct expression of the nucleotide sequence onlyin certain host cells (e.g., tissue-specific regulatory sequences). Itwill be appreciated by those skilled in the art that the design of theexpression vector can depend on such factors as the choice of the hostcell to be transformed, the level of expression of protein desired, andthe like. The expression vectors of the invention can be introduced intohost cells to thereby produce proteins or peptides, including fusionproteins or peptides, encoded by nucleic acids as described herein.

[0198] The recombinant expression vectors of the invention can bedesigned for expression of a marker protein or a segment thereof inprokaryotic (e.g., E. coli) or eukaryotic cells (e.g., insect cells{using baculovirus expression vectors}, yeast cells or mammalian cells).Suitable host cells are discussed further in Goeddel, supra.Alternatively, the recombinant expression vector can be transcribed andtranslated in vitro, for example using T7 promoter regulatory sequencesand T7 polymerase.

[0199] Expression of proteins in prokaryotes is most often carried outin E. coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: 1) to increase expression ofrecombinant protein; 2) to increase the solubility of the recombinantprotein; and 3) to aid in the purification of the recombinant protein byacting as a ligand in affinity purification. Often, in fusion expressionvectors, a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent topurification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith and Johnson, 1988, Gene 67:31-40), pMAL (New England Biolabs,Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuseglutathione S-transferase (GST), maltose E binding protein, or proteinA, respectively, to the target recombinant protein.

[0200] Examples of suitable inducible non-fusion E. coli expressionvectors include pTrc (Amann et al., 1988, Gene 69:301-315) and pET 11d(Studier et al., p. 60-89, In Gene Expression Technology: Methods inEnzymology vol. 1 85, Academic Press, San Diego, Calif., 1991). Targetgene expression from the pTrc vector relies on host RNA polymerasetranscription from a hybrid trp-lac fusion promoter. Target geneexpression from the pET 11d vector relies on transcription from a T7gn10-lac fusion promoter mediated by a co-expressed viral RNA polymerase(T7 gn1). This viral polymerase is supplied by host strains BL21(DE3) orHMS174(DE3) from a resident prophage harboring a T7 gn1 gene under thetranscriptional control of the lacUV 5 promoter.

[0201] One strategy to maximize recombinant protein expression in E.coli is to express the protein in a host bacteria with an impairedcapacity to proteolytically cleave the recombinant protein (Gottesman,p. 119-128, In Gene Expression Technology: Methods in Enzymology vol.185, Academic Press, San Diego, Calif., 1990. Another strategy is toalter the nucleic acid sequence of the nucleic acid to be inserted intoan expression vector so that the individual codons for each amino acidare those preferentially utilized in E. coli (Wada et al., 1992, NucleicAcids Res. 20:2111-2118). Such alteration of nucleic acid sequences ofthe invention can be carried out by standard DNA synthesis techniques.

[0202] In another embodiment, the expression vector is a yeastexpression vector. Examples of vectors for expression in yeast S.cerevisiae include pYepSec1 (Baldari et al., 1987, EMBO J. 6:229-234),pMFa (Kurjan and Herskowitz, 1982, Cell 30:933-943), pJRY88 (Schultz etal., 1987, Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego,Calif.), and pPicZ (Invitrogen Corp, San Diego, Calif.).

[0203] Alternatively, the expression vector is a baculovirus expressionvector. Baculovirus vectors available for expression of proteins incultured insect cells (e.g., Sf 9 cells) include the pAc series (Smithet al., 1983, Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklowand Summers, 1989, Virology 170:31-39).

[0204] In yet another embodiment, a nucleic acid of the invention isexpressed in mammalian cells using a mammalian expression vector.Examples of mammalian expression vectors include pCDM8 (Seed, 1987,Nature 329:840) and pMT2PC (Kaufmnan et al., 1987, EMBO J. 6:187-195).When used in mammalian cells, the expression vector's control functionsare often provided by viral regulatory elements. For example, commonlyused promoters are derived from polyoma, Adenovirus 2, cytomegalovirusand Simian Virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook etal., supra.

[0205] In another embodiment, the recombinant mammalian expressionvector is capable of directing expression of the nucleic acidpreferentially in a particular cell type (e.g., tissue-specificregulatory elements are used to express the nucleic acid).Tissue-specific regulatory elements are known in the art. Non-limitingexamples of suitable tissue-specific promoters include the albuminpromoter (liver-specific; Pinkert et al., 1987, Genes Dev. 1:268-277),lymphoid-specific promoters (Calame and Eaton, 1988, Adv. Immunol.43:235-275), in particular promoters of T cell receptors (Winoto andBaltimore, 1989, EMBO J. 8:729-733) and immunoglobulins (Banedji et al.,1983, Cell 33:729-740; Queen and Baltimore, 1983, Cell 33:741-748),neuron-specific promoters (e.g., the neurofilament promoter; Byrne andRuddle, 1989, Proc. NatL. Acad. Sci. USA 86:5473-5477),pancreas-specific promoters (Edlund et al., 1985, Science 230:912-916),and mammary gland-specific promoters (e.g., milk whey promoter; U.S.Pat. No. 4,873,316 and European Application Publication No. 264,166).Developmentally-regulated promoters are also encompassed, for examplethe murine hox promoters (Kessel and Gruss, 1990, Science 249:374-379)and the α-fetoprotein promoter (Camper and Tilghman, 1989, Genes Dev.3:537-546).

[0206] The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. That is, the DNA molecule isoperably linked to a regulatory sequence in a manner which allows forexpression (by transcription of the DNA molecule) of an RNA moleculewhich is antisense to the mRNA encoding a polypeptide of the invention.Regulatory sequences operably linked to a nucleic acid cloned in theantisense orientation can be chosen which direct the continuousexpression of the antisense RNA molecule in a variety of cell types, forinstance viral promoters and/or enhancers, or regulatory sequences canbe chosen which direct constitutive, tissue-specific or cell typespecific expression of antisense RNA. The antisense expression vectorcan be in the form of a recombinant plasmid, phagemid, or attenuatedvirus in which antisense nucleic acids are produced under the control ofa high efficiency regulatory region, the activity of which can bedetermined by the cell type into which the vector is introduced. For adiscussion of the regulation of gene expression using antisense genessee Weintraub et al., 1986, Trends in Genetics, Vol. 1(1).

[0207] Another aspect of the invention pertains to host cells into whicha recombinant expression vector of the invention has been introduced.The terms “host cell” and “recombinant host cell” are usedinterchangeably herein. It is understood that such terms refer not onlyto the particular subject cell but to the progeny or potential progenyof such a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

[0208] A host cell can be any prokaryotic (e.g., E. coli ) or eukaryoticcell (e.g., insect cells, yeast or mammalian cells).

[0209] Vector DNA can be introduced into prokaryotic or eukaryotic cellsvia conventional transformation or transfection techniques. As usedherein, the terms “transformation” and “transfection” are intended torefer to a variety of art-recognized techniques for introducing foreignnucleic acid into a host cell, including calcium phosphate or calciumchloride co-precipitation, DEAE-dextran-mediated transfection,lipofection, or electroporation. Suitable methods for transforming ortransfecting host cells can be found in Sambrook, et al. (supra), andother laboratory manuals.

[0210] For stable transfection of mammalian cells, it is known that,depending upon the expression vector and transfection technique used,only a small fraction of cells may integrate the foreign DNA into theirgenome. In order to identify and select these integrants, a gene thatencodes a selectable marker (e.g., for resistance to antibiotics) isgenerally introduced into the host cells along with the gene ofinterest. Preferred selectable markers include those which conferresistance to drugs, such as G418, hygromycin and methotrexate. Cellsstably transfected with the introduced nucleic acid can be identified bydrug selection (e.g., cells that have incorporated the selectable markerwill survive, while the other cells die).

[0211] A host cell of the invention, such as a prokaryotic or eukaryotichost cell in culture, can be used to produce a marker protein or asegment thereof. Accordingly, the invention further provides methods forproducing a marker protein or a segment thereof using the host cells ofthe invention. In one embodiment, the method comprises culturing thehost cell of the invention (into which a recombinant expression vectorencoding a marker protein or a segment thereof has been introduced) in asuitable medium such that the is produced. In another embodiment, themethod further comprises isolating the marker protein or a segmentthereof from the medium or the host cell.

[0212] The host cells of the invention can also be used to producenonhuman transgenic animals. For example, in one embodiment, a host cellof the invention is a fertilized oocyte or an embryonic stem cell intowhich a sequences encoding a marker protein or a segment thereof havebeen introduced. Such host cells can then be used to create non-humantransgenic animals in which exogenous sequences encoding a markerprotein of the invention have been introduced into their genome orhomologous recombinant animals in which endogenous gene(s) encoding amarker protein have been altered. Such animals are useful for studyingthe function and/or activity of the marker protein and for identifyingand/or evaluating modulators of marker protein. As used herein, a“transgenic animal” is a non-human animal, preferably a mammal, morepreferably a rodent such as a rat or mouse, in which one or more of thecells of the animal includes a transgene. Other examples of transgenicanimals include non-human primates, sheep, dogs, cows, goats, chickens,amphibians, etc. A transgene is exogenous DNA which is integrated intothe genome of a cell from which a transgenic animal develops and whichremains in the genome of the mature animal, thereby directing theexpression of an encoded gene product in one or more cell types ortissues of the transgenic animal. As used herein, an “homologousrecombinant animal” is a non-human animal, preferably a mammal, morepreferably a mouse, in which an endogenous gene has been altered byhomologous recombination between the endogenous gene and an exogenousDNA molecule introduced into a cell of the animal, e.g., an embryoniccell of the animal, prior to development of the animal.

[0213] A transgenic animal of the invention can be created byintroducing a nucleic acid encoding a marker protein into the malepronuclei of a fertilized oocyte, e.g., by microinjection, retroviralinfection, and allowing the oocyte to develop in a pseudopregnant femalefoster animal. Intronic sequences and polyadenylation signals can alsobe included in the transgene to increase the efficiency of expression ofthe trausgene. A tissue-specific regulatory sequence(s) can be operablylinked to the transgene to direct expression of the polypeptide of theinvention to particular cells. Methods for generating transgenic animalsvia embryo manipulation and microinjection, particularly animals such asmice, have become conventional in the art and are described, forexample, in U.S. Pat. Nos. 4,736,866 and 4,870,009, U.S. Pat. No.4,873,191 and in Hogan, Manipulating the Mouse Embryo, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1986. Similar methodsare used for production of other transgenic animals. A transgenicfounder animal can be identified based upon the presence of thetransgene in its genome and/or expression of mRNA encoding the transgenein tissues or cells of the animals. A transgenic founder animal can thenbe used to breed additional animals carrying the transgene. Moreover,transgenic animals carrying the transgene can fuirther be bred to othertransgenic animals carrying other transgenes.

[0214] To create an homologous recombinant animal, a vector is preparedwhich contains at least a portion of a gene encoding a marker proteininto which a deletion, addition or substitution has been introduced tothereby alter, e~g., functionally disrupt, the gene. In a preferredembodiment, the vector is designed such that, upon homologousrecombination, the endogenous gene is functionally disrupted (i.e., nolonger encodes a functional protein; also referred to as a “knock out”vector). Alternatively, the vector can be designed such that, uponhomologous recombination, the endogenous gene is mutated or otherwisealtered but still encodes functional protein (e.g., the upstreamregulatory region can be altered to thereby alter the expression of theendogenous protein). In the homologous recombination vector, the alteredportion of the gene is flanked at its 5′ and 3′ ends by additionalnucleic acid of the gene to allow for homologous recombination to occurbetween the exogenous gene carried by the vector and an endogenous genein an embryonic stem cell. The additional flanking nucleic acidsequences are of sufficient length for successful homologousrecombination with the endogenous gene. Typically, several kilobases offlanking DNA (both at the 5′ and 3′ ends) are included in the vector(see, e.g., Thomas and Capecchi, 1987, Cell 51:503 for a description ofhomologous recombination vectors). The vector is introduced into anembryonic stem cell line (e.g., by electroporation) and cells in whichthe introduced gene has homologously recombined with the endogenous geneare selected (see, e.g., Li et al., 1992, Cell 69:915). The selectedcells are then injected into a blastocyst of an animal (e.g., a mouse)to form aggregation chimeras (see, e.g., Bradley, Teratocarcinomas andEmbryonic Stem Cells: A Practical Approach, Robertson, Ed., IRL, Oxford,1987, pp. 113-152). A chimeric embryo can then be implanted into asuitable pseudopregnant female foster animal and the embryo brought toterm. Progeny harboring the homologously recombined DNA in their germcells can be used to breed animals in which all cells of the animalcontain the homologously recombined DNA by gernline transmission of thetransgene. Methods for constructing homologous recombination vectors andhomologous recombinant animals are described fuirther in Bradley (1991)Current Opinion in Bio/Technology 2:823-829 and in PCT Publication NOS.WO 90/11354, WO 91/01140, WO 92/0968, and WO 93/04169.

[0215] In another embodiment, transgenic non-human animals can beproduced which contain selected systems which allow for regulatedexpression of the transgene. One example of such a system is thecre/loxP recombinase system of bacteriophage P1. For a description ofthe cre/loxP recombinase system, see, e.g., Lakso et al (1992) Proc.Natl. Acad. Sci. USA 89:6232-6236. Another example of a recombinasesystem is the FLP recombinase system of Saccharomyces cerevisiae(O'Gorman et al., 1991, Science 251:1351-1355). If a cre/loxPrecombinase system is used to regulate expression of the transgene,animals containing transgenes encoding both the Cre recombinase and aselected protein are required. Such animals can be provided through theconstruction of “double” transgenic animals, e.g., by mating twotransgenic animals, one containing a transgene encoding a selectedprotein and the other containing a transgene encoding a recombinase.

[0216] Clones of the non-human transgenic animals described herein canalso be produced according to the methods described in Wilmut et al.(1997) Nature 385:810-813 and PCT Publication NOS. WO 97/07668 and WO97/07669.

[0217] IV. Pharmaceutical Compositions

[0218] The nucleic acid molecules, polypeptides, and antibodies (alsoreferred to herein as “active compounds”) of the invention can beincorporated into pharmaceutical compositions suitable foradministration. Such compositions typically comprise the nucleic acidmolecule, protein, or antibody and a pharmaceutically acceptablecarrier. As used herein the language “pharmaceutically acceptablecarrier” is intended to include any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like, compatible with pharmaceuticaladministration. The use of such media and agents for pharmaceuticallyactive substances is well known in the art. Except insofar as anyconventional media or agent is incompatible with the active compound,use thereof in the compositions is contemplated. Supplementary activecompounds can also be incorporated into the compositions.

[0219] The invention includes methods for preparing pharmaceuticalcompositions for modulating the expression or activity of a markernucleic acid or protein . Such methods comprise formulating apharmaceutically acceptable carrier with an agent which modulatesexpression or activity of a marker nucleic acid or protein. Suchcompositions can further include additional active agents. Thus, theinvention further includes methods for preparing a pharmaceuticalcomposition by formulating a pharmaceutically acceptable carrier with anagent which modulates expression or activity of a marker nucleic acid orprotein and one or more additional active compounds.

[0220] The invention also provides methods (also referred to herein as“screening assays”) for identifying modulators, i.e., candidate or testcompounds or agents (e.g., peptides, peptidomimetics, peptoids, smallmolecules or other drugs) which (a) bind to the marker, or (b) have amodulatory (e.g., stimulatory or inhibitory) effect on the activity ofthe marker or, more specifically, (c) have a modulatory effect on theinteractions of the marker with one or more of its natural substrates(e.g., peptide, protein, hormone, co-factor, or nucleic acid), or (d)have a modulatory effect on the expression of the marker. Such assaystypically comprise a reaction between the marker and one or more assaycomponents. The other components may be either the test compound itself,or a combination of test compound and a natural binding partner of themarker.

[0221] The test compounds of the present invention may be obtained fromany available source, including systematic libraries of natural and/orsynthetic compounds. Test compounds may also be obtained by any of thenumerous approaches in combinatorial library methods known in the art,including: biological libraries; peptoid libraries (libraries ofmolecules having the finctionalities of peptides, but with a novel,non-peptide backbone which are resistant to enzymatic degradation butwhich nevertheless remain bioactive; see, e.g., Zuckermann et aL, 1994,J. Med. Chem. 37:2678-85); spatially addressable parallel solid phase orsolution phase libraries; synthetic library methods requiringdeconvolution; the ‘one-bead one-compound’ library method; and syntheticlibrary methods using affinity chromatography selection. The biologicallibrary and peptoid library approaches are limited to peptide libraries,while the other four approaches are applicable to peptide, non-peptideoligomer or small molecule libraries of compounds (Lam, 1997, AnticancerDrug Des. 12:145).

[0222] Examples of methods for the synthesis of molecular libraries canbe found in the art, for example in: DeWitt et al. (1993) Proc. Natl.Acad. Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al.(1993) Science 261:1303; Carrell et aL (1994) Angew. Chem. Int. Ed.Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061;and in Gallop et al. (1994) J. Med. Chem. 37:1233.

[0223] Libraries of compounds may be presented in solution (e.g.,Houghten, 1992, Biotechniques 13:412-421), or on beads (Lam, 1991,Nature 354:82-84), chips (Fodor, 1993, Nature 364:555-556), bacteriaand/or spores, (Ladner, U.S. Pat. No. 5,223,409), plasmids (Cull et al,1992, Proc Natl Acad Sci USA 89:1865-1869) or on phage (Scott and Smith,1990, Science 249:386-390; Devlin, 1990, Science 249:404-406; Cwirla etal, 1990, Proc. Natl. Acad. Sci. 87:6378-6382; Felici, 1991, J. Mol.Biol. 222:301-310; Ladner, supra.).

[0224] In one embodiment, the invention provides assays for screeningcandidate or test compounds which are substrates of a protein encoded byor corresponding to a marker or biologically active portion thereof. Inanother embodiment, the invention provides assays for screeningcandidate or test compounds which bind to a protein encoded by orcorresponding to a marker or biologically active portion thereof.Determining the ability of the test compound to directly bind to aprotein can be accomplished, for example, by coupling the compound witha radioisotope or enzymatic label such that binding of the compound tothe marker can be determined by detecting the labeled marker compound ina complex. For example, compounds (e.g., marker substrates) can belabeled with ¹²⁵I, ³⁵S, ¹⁴C, or ³H, either directly or indirectly, andthe radioisotope detected by direct counting of radioemission or byscintillation counting. Alternatively, assay components can beenzymatically labeled with, for example, horseradish peroxidase,alkaline phosphatase, or luciferase, and the enzymatic label detected bydetermination of conversion of an appropriate substrate to product.

[0225] In another embodiment, the invention provides assays forscreening candidate or test compounds which modulate the expression of amarker or the activity of a protein encoded by or corresponding to amarker, or a biologically active portion thereof. In all likelihood, theprotein encoded by or corresponding to the marker can, in vivo, interactwith one or more molecules, such as but not limited to, peptides,proteins, hormones, cofactors and nucleic acids. For the purposes ofthis discussion, such cellular and extracellular molecules are referredto herein as “binding partners” or marker “substrate”.

[0226] One necessary embodiment of the invention in order to facilitatesuch screening is the use of a protein encoded by or corresponding tomarker to identify the protein's natural in vivo binding partners. Thereare many ways to accomplish this which are known to one skilled in theart. One example is the use of the marker protein as “bait protein” in atwo-hybrid assay or three-hybrid assay (see, e.g., U.S. Pat. No.5,283,317; Zervos et al, 1993, Cell 72:223-232; Madura et al, 1993, J.Biol. Chem. 268:12046-12054; Bartel et al, 1993, Biotechniques14:920-924; Iwabuchi et al, 1993 Oncogene 8:1693-1696; Brent WO94/10300)in order to identify other proteins which bind to or interact with themarker (binding partners) and, therefore, are possibly involved in thenatural finction of the marker. Such marker binding partners are alsolikely to be involved in the propagation of signals by the markerprotein or downstream elements of a marker protein-mediated signalingpathway. Alternatively, such marker protein binding partners may also befound to be inhibitors of the marker protein.

[0227] The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that encodes a marker proteinfused to a gene encoding the DNA binding domain of a known transcriptionfactor (e.g., GAL-4). In the other construct, a DNA sequence, from alibrary of DNA sequences, that encodes an unidentified protein (“prey”or “sample”) is fused to a gene that codes for the activation domain ofthe known transcription factor. If the “bait” and the “prey” proteinsare able to interact, in vivo, forming a marker-dependent complex, theDNA-binding and activation domains of the transcription factor arebrought into close proximity. This proximity allows transcription of areporter gene (e.g., LacZ) which is operably linked to a transcriptionalregulatory site responsive to the transcription factor. Expression ofthe reporter gene can be readily detected and cell colonies containingthe functional transcription factor can be isolated and used to obtainthe cloned gene which encodes the protein which interacts with themarker protein.

[0228] In a further embodiment, assays may be devised through the use ofthe invention for the purpose of identifying compounds which modulate(e.g., affect either positively or negatively) interactions between amarker protein and its substrates and/or binding partners. Suchcompounds can include, but are not limited to, molecules such asantibodies, peptides, hormones, oligonucleotides, nucleic acids, andanalogs thereof. Such compounds may also be obtained from any availablesource, including systematic libraries of natural and/or syntheticcompounds. The preferred assay components for use in this embodiment isan cervical cancer marker protein identified herein, the known bindingpartner and/or substrate of same, and the test compound. Test compoundscan be supplied from any source.

[0229] The basic principle of the assay systems used to identifycompounds that interfere with the interaction between the marker proteinand its binding partner involves preparing a reaction mixture containingthe marker protein and its binding partner under conditions and for atime sufficient to allow the two products to interact and bind, thusforming a complex. In order to test an agent for inhibitory activity,the reaction mixture is prepared in the presence and absence of the testcompound. The test compound can be initially included in the reactionmixture, or can be added at a time subsequent to the addition of themarker protein and its binding partner. Control reaction mixtures areincubated without the test compound or with a placebo. The formation ofany complexes between the marker protein and its binding partner is thendetected. The formation of a complex in the control reaction, but lessor no such formation in the reaction mixture containing the testcompound, indicates that the compound interferes with the interaction ofthe marker protein and its binding partner. Conversely, the formation ofmore complex in the presence of compound than in the control reactionindicates that the compound may enhance interaction of the markerprotein and its binding partner.

[0230] The assay for compounds that interfere with the interaction ofthe marker protein with its binding partner may be conducted in aheterogeneous or homogeneous format. Heterogeneous assays involveanchoring either the marker protein or its binding partner onto a solidphase and detecting complexes anchored to the solid phase at the end ofthe reaction. In homogeneous assays, the entire reaction is carried outin a liquid phase. In either approach, the order of addition ofreactants can be varied to obtain different information about thecompounds being tested. For example, test compounds that interfere withthe interaction between the marker proteins and the binding partners(e.g., by competition) can be identified by conducting the reaction inthe presence of the test substance, i.e., by adding the test substanceto the reaction mixture prior to or simultaneously with the marker andits interactive binding partner. Alternatively, test compounds thatdisrupt preformed complexes, e.g., compounds with higher bindingconstants that displace one of the components from the complex, can betested by adding the test compound to the reaction mixture aftercomplexes have been formed. The various formats are briefly describedbelow.

[0231] In a heterogeneous assay system, either the marker protein or itsbinding partner is anchored onto a solid surface or matrix, while theother corresponding non-anchored component may be labeled, eitherdirectly or indirectly. In practice, microtitre plates are oftenutilized for this approach. The anchored species can be immobilized by anumber of methods, either non-covalent or covalent, that are typicallywell known to one who practices the art. Non-covalent attachment canoften be accomplished simply by coating the solid surface with asolution of the marker protein or its binding partner and drying.Alternatively, an immobilized antibody specific for the assay componentto be anchored can be used for this purpose. Such surfaces can often beprepared in advance and stored.

[0232] In related embodiments, a fusion protein can be provided whichadds a domain that allows one or both of the assay components to beanchored to a matrix. For example, glutathione-S-transferase/markerfusion proteins or glutathione-S-transferase/binding partner can beadsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis,Mo.) or glutathione derivatized microtiter plates, which are thencombined with the test compound or the test compound and either thenon-adsorbed marker or its binding partner, and the mixture incubatedunder conditions conducive to complex formation (e.g., physiologicalconditions). Following incubation, the beads or microtiter plate wellsare washed to remove any unbound assay components, the immobilizedcomplex assessed either directly or indirectly, for example, asdescribed above. Alternatively, the complexes can be dissociated fromthe matrix, and the level of marker binding or activity determined usingstandard techniques.

[0233] Other techniques for immobilizing proteins on matrices can alsobe used in the screening assays of the invention. For example, either amarker protein or a marker protein binding partner can be immobilizedutilizing conjugation of biotin and streptavidin. Biotinylated markerprotein or target molecules can be prepared from biotin-NHS(N-hydroxy-succinimide) using techniques known in the art (e.g.,biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized inthe wells of streptavidin-coated 96 well plates (Pierce Chemical). Incertain embodiments, the protein-immobilized surfaces can be prepared inadvance and stored.

[0234] In order to conduct the assay, the corresponding partner of theimmobilized assay component is exposed to the coated surface with orwithout the test compound. After the reaction is complete, unreactedassay components are removed (e.g., by washing) and any complexes formedwill remain immobilized on the solid surface. The detection of complexesanchored on the solid surface can be accomplished in a number of ways.Where the non-immobilized component is pre-labeled, the detection oflabel immobilized on the surface indicates that complexes were formed.Where the non-immobilized component is not pre-labeled, an indirectlabel can be used to detect complexes anchored on the surface; e.g.,using a labeled antibody specific for the initially non-immobilizedspecies (the antibody, in turn, can be directly labeled or indirectlylabeled with, e.g., a labeled anti-Ig antibody). Depending upon theorder of addition of reaction components, test compounds which modulate(inhibit or enhance) complex formation or which disrupt preformedcomplexes can be detected.

[0235] In an alternate embodiment of the invention, a homogeneous assaymay be used. This is typically a reaction, analogous to those mentionedabove, which is conducted in a liquid phase in the presence or absenceof the test compound. The formed complexes are then separated fromunreacted components, and the amount of complex formed is determined. Asmentioned for heterogeneous assay systems, the order of addition ofreactants to the liquid phase can yield information about which testcompounds modulate (inhibit or enhance) complex formation and whichdisrupt preformed complexes.

[0236] In such a homogeneous assay, the reaction products may beseparated from unreacted assay components by any of a number of standardtechniques, including but not limited to: differential centrifugation,chromatography, electrophoresis and immunoprecipitation. In differentialcentrifugation, complexes of molecules may be separated from uncomplexedmolecules through a series of centrifugal steps, due to the differentsedimentation equilibria of complexes based on their different sizes anddensities (see, for example, Rivas, G., and Minton, A. P., TrendsBiochem Sci 1993 Aug;18(8):284-7). Standard chromatographic techniquesmay also be utilized to separate complexed molecules from uncomplexedones. For example, gel filtration chromatography separates moleculesbased on size, and through the utilization of an appropriate gelfiltration resin in a column format, for example, the relatively largercomplex may be separated from the relatively smaller uncomplexedcomponents. Similarly, the relatively different charge properties of thecomplex as compared to the uncomplexed molecules may be exploited todifferentially separate the complex from the remaining individualreactants, for example through the use of ion-exchange chromatographyresins. Such resins and chromatographic techniques are well known to oneskilled in the art (see, e.g., Heegaard, 1998, J. Mol. Recognit.11:141-148; Hage and Tweed, 1997, J. Chromatogr. B. Biomed. Sci. Appl.,699:499-525). Gel electrophoresis may also be employed to separatecomplexed molecules from unbound species (see, e.g., Ausubel et al(eds.), In: Current Protocols in Molecular Biology, J. Wiley & Sons, NewYork. 1999). In this technique, protein or nucleic acid complexes areseparated based on size or charge, for example. In order to maintain thebinding interaction during the electrophoretic process, nondenaturinggels in the absence of reducing agent are typically preferred, butconditions appropriate to the particular interactants will be well knownto one skilled in the art. Immunoprecipitation is another commontechnique utilized for the isolation of a protein-protein complex fromsolution (see, e.g., Ausubel et al (eds.), In: Current Protocols inMolecular Biology, J. Wiley & Sons, New York. 1999). In this technique,all proteins binding to an antibody specific to one of the bindingmolecules are precipitated from solution by conjugating the antibody toa polymer bead that may be readily collected by centrifugation. Thebound assay components are released from the beads (through a specificproteolysis event or other technique well known in the art which willnot disturb the protein-protein interaction in the complex), and asecond immunoprecipitation step is performed, this time utilizingantibodies specific for the correspondingly different interacting assaycomponent. In this manner, only formed complexes should remain attachedto the beads. Variations in complex formation in both the presence andthe absence of a test compound can be compared, thus offeringinformation about the ability of the compound to modulate interactionsbetween the marker protein and its binding partner.

[0237] Also within the scope of the present invention are methods fordirect detection of interactions between the marker protein and itsnatural binding partner and/or a test compound in a homogeneous orheterogeneous assay system without further sample manipulation. Forexample, the technique of fluorescence energy transfer may be utilized(see, e.g., Lakowicz et al, U.S. Pat. No. 5,631,169; Stavrianopoulos etal, U.S. Pat. No. 4,868,103). Generally, this technique involves theaddition of a fluorophore label on a first ‘donor’ molecule (e.g.,marker or test compound) such that its emitted fluorescent energy willbe absorbed by a fluorescent label on a second, ‘acceptor’ molecule(e.g., marker or test compound), which in turn is able to fluoresce dueto the absorbed energy. Alternately, the ‘donor’ protein molecule maysimply utilize the natural fluorescent energy of tryptophan residues.Labels are chosen that emit different wavelengths of light, such thatthe ‘acceptor’ molecule label may be differentiated from that of the‘donor’. Since the efficiency of energy transfer between the labels isrelated to the distance separating the molecules, spatial relationshipsbetween the molecules can be assessed. In a situation in which bindingoccurs between the molecules, the fluorescent emission of the ‘acceptor’molecule label in the assay should be maximal. An FET binding event canbe conveniently measured through standard fluorometric detection meanswell known in the art (e.g., using a fluorimeter). A test substancewhich either enhances or hinders participation of one of the species inthe preformed complex will result in the generation of a signal variantto that of background. In this way, test substances that modulateinteractions between a marker and its binding partner can be identifiedin controlled assays.

[0238] In another embodiment, modulators of marker expression areidentified in a method wherein a cell is contacted with a candidatecompound and the expression of marker mRNA or protein in the cell, isdetermined. The level of expression of marker MRNA or protein in thepresence of the candidate compound is compared to the level ofexpression of marker MRNA or protein in the absence of the candidatecompound. The candidate compound can then be identified as a modulatorof marker expression based on this comparison. For example, whenexpression of marker MRNA or protein is greater (statisticallysignificantly greater) in the presence of the candidate compound than inits absence, the candidate compound is identified as a stimulator ofmarker mRNA or protein expression. Conversely, when expression of markermRNA or protein is less (statistically significantly less) in thepresence of the candidate compound than in its absence, the candidatecompound is identified as an inhibitor of marker mRNA or proteinexpression. The level of marker MRNA or protein expression in the cellscan be determined by methods described herein for detecting marker mRNAor protein.

[0239] In another aspect, the invention pertains to a combination of twoor more of the assays described herein. For example, a modulating agentcan be identified using a cell-based or a cell free assay, and theability of the agent to modulate the activity of a marker protein can befurther confirmed in vivo, e.g., in a whole animal model for cellulartransformation and/or tumorigenesis.

[0240] This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein inan appropriate animal model. For example, an agent identified asdescribed herein (e.g., a marker modulating agent, an antisense markernucleic acid molecule, a marker-specific antibody, or a marker-bindingpartner) can be used in an animal model to determine the efficacy,toxicity, or side effects of treatment with such an agent.Alternatively, an agent identified as described herein can be used in ananimal model to determine the mechanism of action of such an agent.Furthermore, this invention pertains to uses of novel agents identifiedby the above-described screening assays for treatments as describedherein.

[0241] It is understood that appropriate doses of small molecule agentsand protein or polypeptide agents depends upon a number of factorswithin the knowledge of the ordinarily skilled physician, veterinarian,or researcher. The dose(s) of these agents will vary, for example,depending upon the identity, size, and condition of the subject orsample being treated, further depending upon the route by which thecomposition is to be administered, if applicable, and the effect whichthe practitioner desires the agent to have upon the nucleic acid orpolypeptide of the invention. Exemplary doses of a small moleculeinclude milligram or microgram amounts per kilogram of subject or sampleweight (e.g. about 1 microgram per kilogram to about 500 milligrams perkilogram, about 100 micrograms per kilogram to about 5 milligrams perkilogram, or about 1 microgram per kilogram to about 50 micrograms perkilogram). Exemplary doses of a protein or polypeptide include gram,milligram or microgram amounts per kilogram of subject or sample weight(e.g. about 1 microgram per kilogram to about 5 grams per kilogram,about 100 micrograms per kilogram to about 500 milligrams per kilogram,or about 1 milligram per kilogram to about 50 milligrams per kilogram).It is furthermore understood that appropriate doses of one of theseagents depend upon the potency of the agent with respect to theexpression or activity to be modulated. Such appropriate doses can bedetermined using the assays described herein. When one or more of theseagents is to be administered to an animal (e.g. a human) in order tomodulate expression or activity of a polypeptide or nucleic acid of theinvention, a physician, veterinarian, or researcher can, for example,prescribe a relatively low dose at first, subsequently increasing thedose until an appropriate response is obtained. In addition, it isunderstood that the specific dose level for any particular animalsubject will depend upon a variety of factors including the activity ofthe specific agent employed, the age, body weight, general health,gender, and diet of the subject, the time of administration, the routeof administration, the rate of excretion, any drug combination, and thedegree of expression or activity to be modulated.

[0242] A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediamine-tetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampules,disposable syringes or multiple dose vials made of glass or plastic.

[0243] Pharmaceutical compositions suitable for injectable use includesterile aqueous solutions (where water soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersions. For vintravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL (BASF; Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifingal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride inthe composition. Prolonged absorption of the injectable compositions canbe brought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

[0244] Sterile injectable solutions can be prepared by incorporating theactive compound (e.g., a polypeptide or antibody) in the required amountin an appropriate solvent with one or a combination of ingredientsenumerated above, as required, followed by filtered sterilization.Generally, dispersions are prepared by incorporating the active compoundinto a sterile vehicle which contains a basic dispersion medium, andthen incorporating the required other ingredients from those enumeratedabove. In the case of sterile powders for the preparation of sterileinjectable solutions, the preferred methods of preparation are vacuumdrying and freeze-drying which yields a powder of the active ingredientplus any additional desired ingredient from a previouslysterile-filtered solution thereof.

[0245] Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed.

[0246] Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches, and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

[0247] For administration by inhalation, the compounds are delivered inthe form of an aerosol spray from a pressurized container or dispenserwhich contains a suitable propellant, e.g., a gas such as carbondioxide, or a nebulizer.

[0248] Systemic administration can also be by transmucosal ortransdermal means. For transmucosal or transdermal administration,penetrants appropriate to the barrier to be permeated are used in theformulation. Such penetrants are generally known in the art, andinclude, for example, for transmucosal administration, detergents, bilesalts, and fusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

[0249] The compounds can also be prepared in the form of suppositories(e.g., with conventional suppository bases such as cocoa butter andother glycerides) or retention enemas for rectal delivery.

[0250] In one embodiment, the active compounds are prepared withcarriers that will protect the compound against rapid elimination fromthe body, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes having monoclonal antibodies incorporated thereinor thereon) can also be used as pharmaceutically acceptable carriers.These can be prepared according to methods known to those skilled in theart, for example, as described in U.S. Pat. No. 4,522,811.

[0251] It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

[0252] For antibodies, the preferred dosage is 0.1 mg/kg to 100 mg/kg ofbody weight (generally 10 mg/kg to 20 mg/kg). If the antibody is to actin the brain, a dosage of 50 mg/kg to 100 mg/kg is usually appropriate.Generally, partially human antibodies and fully human antibodies have alonger half-life within the human body than other antibodies.Accordingly, lower dosages and less frequent administration is oftenpossible. Modifications such as lipidation can be used to stabilizeantibodies and to enhance uptake and tissue penetration (e.g., into thecervical epithelium). A method for lipidation of antibodies is describedby Cruikshank et al. (1997) J. Acquired Immune Deficiency Syndromes andHuman Retrovirology 14:193.

[0253] The invention also provides vaccine compositions for theprevention and/or treatment of cervical cancer. The invention providescervical cancer vaccine compositions in which a protein of a marker ofTable 1, or a combination of proteins of the markers of Table 1, areintroduced into a subject in order to stimulate an immune responseagainst the cervical cancer. The invention also provides cervical cancervaccine compositions in which a gene expression construct, whichexpresses a marker or fragment of a marker identified in Table 1, isintroduced into the subject such that a protein or fragment of a proteinencoded by a marker of Table 1 is produced by transfected cells in thesubject at a higher than normal level and elicits an immune response.

[0254] In one embodiment, a cervical cancer vaccine is provided andemployed as an immunotherapeutic agent for the prevention of cervicalcancer. In another embodiment, a cervical cancer vaccine is provided andemployed as an immunotherapeutic agent for the treatment of cervicalcancer.

[0255] By way of example, a cervical cancer vaccine comprised of theproteins of the markers of Table 1, may be employed for the preventionand/or treatment of cervical cancer in a subject by administering thevaccine by a variety of routes, e.g., intradermally, subcutaneously, orintramuscularly. In addition, the cervical cancer vaccine can beadministered together with adjuvants and/or immunomodulators to boostthe activity of the vaccine and the subject's response. In oneembodiment, devices and/or compositions containing the vaccine, suitablefor sustained or intermittent release could be, implanted in the body ortopically applied thereto for the relatively slow release of suchmaterials into the body. The cervical cancer vaccine can be introducedalong with immunomodulatory compounds, which can alter the type ofimmune response produced in order to produce a response which will bemore effective in eliminating the cancer.

[0256] In another embodiment, a cervical cancer vaccine comprised of anexpression construct of the markers of Table 1, may be introduced byinjection into muscle or by coating onto microprojectiles and using adevice designed for the purpose to fire the projectiles at high speedinto the skin. The cells of the subject will then express the protein(s)or fragments of proteins of the markers of Table 1 and induce an immuneresponse. In addition, the cervical cancer vaccine may be introducedalong with expression constructs for immunomodulatory molecules, such ascytokines, which may increase the immune response or modulate the typeof immune response produced in order to produce a response which will bemore effective in eliminating the cancer.

[0257] The marker nucleic acid molecules can be inserted into vectorsand used as gene therapy vectors. Gene therapy vectors can be deliveredto a subject by, for example, intravenous injection, localadministration (U.S. Pat. No. 5,328,470), or by stereotactic injection(see, e.g., Chen et aL, 1994, Proc. Natl. Acad. Sci. USA 91:3054-3057).The pharmaceutical preparation of the gene therapy vector can includethe gene therapy vector in an acceptable diluent, or can comprise a slowrelease matrix in which the gene delivery vehicle is imbedded.Alternatively, where the complete gene delivery vector can be producedintact from recombinant cells, e.g. retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system.

[0258] The pharmaceutical compositions can be included in a container,pack, or dispenser together with instructions for administration.

[0259] V. Predictive Medicine

[0260] The present invention pertains to the field of predictivemedicine in which diagnostic assays, prognostic assays,pharmacogenomics, and monitoring clinical trails are used for prognostic(predictive) purposes to thereby treat an individual prophylactically.Accordingly, one aspect of the present invention relates to diagnosticassays for determining the level of expression of one or more markerproteins or nucleic acids, in order to determine whether an individualis at risk of developing cervical cancer. Such assays can be used forprognostic or predictive purposes to thereby prophylactically treat anindividual prior to the onset of the cancer.

[0261] Yet another aspect of the invention pertains to monitoring theinfluence of agents (e.g., drugs or other compounds administered eitherto inhibit cervical cancer or to treat or prevent any other disorder{i.e. in order to understand any cervical carcinogenic effects that suchtreatment may have}) on the expression or activity of a marker of theinvention in clinical trials. These and other agents are described infurther detail in the following sections.

[0262] A. Diagnostic Assays

[0263] An exemplary method for detecting the presence or absence of amarker protein or nucleic acid in a biological sample involves obtaininga biological sample (e.g. a cervical-associated body fluid) from a testsubject and contacting the biological sample with a compound or an agentcapable of detecting the polypeptide or nucleic acid (e.g., mRNA,genomic DNA, or cDNA). The detection methods of the invention can thusbe used to detect mRNA, protein, cDNA, or genomic DNA, for example, in abiological sample in vitro as well as in vivo. For example, in vitrotechniques for detection of mRNA include Northern hybridizations and insitu hybridizations. In vitro techniques for detection of a markerprotein include enzyme linked immunosorbent assays (ELISAs), Westernblots, immunoprecipitations and immunofluorescence. In vitro techniquesfor detection of genomic DNA include Southern hybridizations.Furthermore, in vivo techniques for detection of a marker proteininclude introducing into a subject a labeled antibody directed againstthe protein or fragment thereof. For example, the antibody can belabeled with a radioactive marker whose presence and location in asubject can be detected by standard imaging techniques.

[0264] A general principle of such diagnostic and prognostic assaysinvolves preparing a sample or reaction mixture that may contain amarker, and a probe, under appropriate conditions and for a timesufficient to allow the marker and probe to interact and bind, thusforming a complex that can be removed and/or detected in the reactionmixture. These assays can be conducted in a variety of ways.

[0265] For example, one method to conduct such an assay would involveanchoring the marker or probe onto a solid phase support, also referredto as a substrate, and detecting target marker/probe complexes anchoredon the solid phase at the end of the reaction. In one embodiment of sucha method, a sample from a subject, which is to be assayed for presenceand/or concentration of marker, can be anchored onto a carrier or solidphase support. In another embodiment, the reverse situation is possible,in which the probe can be anchored to a solid phase and a sample from asubject can be allowed to react as an unanchored component of the assay.

[0266] There are many established methods for anchoring assay componentsto a solid phase. These include, without limitation, marker or probemolecules which are immobilized through conjugation of biotin andstreptavidin. Such biotinylated assay components can be prepared frombiotin-NHS (N-hydroxy-succinimide) using techniques known in the art(e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), andimmobilized in the wells of streptavidin-coated 96 well plates (PierceChemical). In certain embodiments, the surfaces with immobilized assaycomponents can be prepared in advance and stored.

[0267] Other suitable carriers or solid phase supports for such assaysinclude any material capable of binding the class of molecule to whichthe marker or probe belongs. Well-known supports or carriers include,but are not limited to, glass, polystyrene, nylon, polypropylene, nylon,polyethylene, dextran, amylases, natural and modified celluloses,polyacrylamides, gabbros, and magnetite.

[0268] In order to conduct assays with the above mentioned approaches,the non-immobilized component is added to the solid phase upon which thesecond component is anchored. After the reaction is complete,uncomplexed components may be removed (e.g., by washing) underconditions such that any complexes formed will remain immobilized uponthe solid phase. The detection of marker/probe complexes anchored to thesolid phase can be accomplished in a number of methods outlined herein.

[0269] In a preferred embodiment, the probe, when it is the unanchoredassay component, can be labeled for the purpose of detection and readoutof the assay, either directly or indirectly, with detectable labelsdiscussed herein and which are well-known to one skilled in the art.

[0270] It is also possible to directly detect marker/probe complexformation without fuirther manipulation or labeling of either component(marker or probe), for example by utilizing the technique offluorescence energy transfer (see, for example, Lakowicz et al., U.S.Pat. No. 5,631,169; Stavrianopoulos, et al., U.S. Pat. No. 4,868,103). Afluorophore label on the first, ‘donor’ molecule is selected such that,upon excitation with incident light of appropriate wavelength, itsemitted fluorescent energy will be absorbed by a fluorescent label on asecond ‘acceptor’ molecule, which in turn is able to fluoresce due tothe absorbed energy. Alternately, the ‘donor’ protein molecule maysimply utilize the natural fluorescent energy of tryptophan residues.Labels are chosen that emit different wavelengths of light, such thatthe ‘acceptor’ molecule label may be differentiated from that of the‘donor’. Since the efficiency of energy transfer between the labels isrelated to the distance separating the molecules, spatial relationshipsbetween the molecules can be assessed. In a situation in which bindingoccurs between the molecules, the fluorescent emission of the ‘acceptor’molecule label in the assay should be maximal. An FET binding event canbe conveniently measured through standard fluorometric detection meanswell known in the art (e.g., using a fluorimeter).

[0271] In another embodiment, determination of the ability of a probe torecognize a marker can be accomplished without labeling either assaycomponent (probe or marker) by utilizing a technology such as real-timeBiomolecular Interaction Analysis (BIA) (see, e.g., Sjolander, S. andUrbaniczky, C., 1991, Anal Chem. 63:2338-2345 and Szabo et al., 1995,Curr. Opin. Struct. Biol. 5:699-705). As used herein, “BIA” or “surfaceplasmon resonance” is a technology for studying biospecific interactionsin real time, without labeling any of the interactants (e.g., BIAcore).Changes in the mass at the binding surface (indicative of a bindingevent) result in alterations of the refractive index of light near thesurface (the optical phenomenon of surface plasmon resonance (SPR)),resulting in a detectable signal which can be used as an indication ofreal-time reactions between biological molecules.

[0272] Alternatively, in another embodiment, analogous diagnostic andprognostic assays can be conducted with marker and probe as solutes in aliquid phase. In such an assay, the complexed marker and probe areseparated from uncomplexed components by any of a number of standardtechniques, including but not limited to: differential centrifugation,chromatography, electrophoresis and immunoprecipitation. In differentialcentrifugation, marker/probe complexes may be separated from uncomplexedassay components through a series of centrifugal steps, due to thedifferent sedimentation equilibria of complexes based on their differentsizes and densities (see, for example, Rivas, G., and Minton, A. P.,1993, Trends Biochem Sci. 18(8):284-7). Standard chromatographictechniques may also be utilized to separate complexed molecules fromuncomplexed ones. For example, gel filtration chromatography separatesmolecules based on size, and through the utilization of an appropriategel filtration resin in a column format, for example, the relativelylarger complex may be separated from the relatively smaller uncomplexedcomponents. Similarly, the relatively different charge properties of themarker/probe complex as compared to the uncomplexed components may beexploited to differentiate the complex from uncomplexed components, forexample through the utilization of ion-exchange chromatography resins.Such resins and chromatographic techniques are well known to one skilledin the art (see, e.g., Heegaard, N. H., 1998, J. Mol. Recognit. Winter11(1-6):141-8; Hage, D. S., and Tweed, S. A. J Chromatogr B Biomed SciAppl October 1997 10;699(1-2):499-525). Gel electrophoresis may also beemployed to separate complexed assay components from unbound components(see, e.g., Ausubel et al., ed., Current Protocols in Molecular Biology,John Wiley & Sons, New York, 1987-1999). In this technique, protein ornucleic acid complexes are separated based on size or charge, forexample. In order to maintain the binding interaction during theelectrophoretic process, non-denaturing gel matrix materials andconditions in the absence of reducing agent are typically preferred.Appropriate conditions to the particular assay and components thereofwill be well known to one skilled in the art.

[0273] In a particular embodiment, the level of marker mRNA can bedetermined both by in situ and by in vitro formats in a biologicalsample using methods known in the art. The term “biological sample” isintended to include tissues, cells, biological fluids and isolatesthereof, isolated from a subject, as well as tissues, cells and fluidspresent within a subject. Many expression detection methods use isolatedRNA. For in vitro methods, any RNA isolation technique that does notselect against the isolation of MRNA can be utilized for thepurification of RNA from cervical cells (see, e.g., Ausubel et al., ed.,Current Protocols in Molecular Biology, John Wiley & Sons, New York1987-1999). Additionally, large numbers of tissue samples can readily beprocessed using techniques well known to those of skill in the art, suchas, for example, the single-step RNA isolation process of Chomczynski(1989, U.S. Pat. No. 4,843,155).

[0274] The isolated MRNA can be used in hybridization or amplificationassays that include, but are not limited to, Southern or Northernanalyses, polymerase chain reaction analyses and probe arrays. Onepreferred diagnostic method for the detection of mRNA levels involvescontacting the isolated mRNA with a nucleic acid molecule (probe) thatcan hybridize to the MRNA encoded by the gene being detected. Thenucleic acid probe can be, for example, a full-length cDNA, or a portionthereof, such as an oligonucleotide of at least 7, 15, 30, 50, 100, 250or 500 nucleotides in length and sufficient to specifically hybridizeunder stringent conditions to a mRNA or genomic DNA encoding a marker ofthe present invention. Other suitable probes for use in the diagnosticassays of the invention are described herein. Hybridization of an mRNAwith the probe indicates that the marker in question is being expressed.

[0275] In one format, the MRNA is immobilized on a solid surface andcontacted with a probe, for example by running the isolated mRNA on anagarose gel and transferring the mRNA from the gel to a membrane, suchas nitrocellulose. In an alternative format, the probe(s) areimmobilized on a solid surface and the mRNA is contacted with theprobe(s), for example, in an Affymetrix gene chip array. A skilledartisan can readily adapt known mRNA detection methods for use indetecting the level of mRNA encoded by the markers of the presentinvention.

[0276] An alternative method for determining the level of MRNA marker ina sample involves the process of nucleic acid amplification, e.g., byrtPCR (the experimental embodiment set forth in Mullis, 1987, U.S. Pat.No. 4,683,202), ligase chain reaction (Barany, 1991, Proc. Natl. Acad.Sci. USA, 88:189-193), self sustained sequence replication (Guatelli etal., 1990, Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptionalamplification system (Kwoh et al., 1989, Proc. Natl. Acad. Sci. USA86:1173-1177), Q-Beta Replicase (Lizardi et al., 1988, Bio/Technology6:1197), rolling circle replication (Lizardi et al., U.S. Pat. No.5,854,033) or any other nucleic acid amplification method, followed bythe detection of the amplified molecules using techniques well known tothose of skill in the art. These detection schemes are especially usefulfor the detection of nucleic acid molecules if such molecules arepresent in very low numbers. As used herein, amplification primers aredefined as being a pair of nucleic acid molecules that can anneal to 5′or 3′ regions of a gene (plus and minus strands, respectively, orvice-versa) and contain a short region in between. In general,amplification primers are from about 10 to 30 nucleotides in length andflank a region from about 50 to 200 nucleotides in length. Underappropriate conditions and with appropriate reagents, such primerspermit the amplification of a nucleic acid molecule comprising thenucleotide sequence flanked by the primers.

[0277] For in situ methods, MRNA does not need to be isolated from thecervical cells prior to detection. In such methods, a cell or tissuesample is prepared/processed using known histological methods. Thesample is then immobilized on a support, typically a glass slide, andthen contacted with a probe that can hybridize to mRNA that encodes themarker.

[0278] As an alternative to making determinations based on the absoluteexpression level of the marker, determinations may be based on thenormalized expression level of the marker. Expression levels arenormalized by correcting the absolute expression level of a marker bycomparing its expression to the expression of a gene that is not amarker, e.g., a housekeeping gene that is constitutively expressed.Suitable genes for normalization include housekeeping genes such as theactin gene, or epithelial cell-specific genes. This normalization allowsthe comparison of the expression level in one sample, e.g., a patientsample, to another sample, e.g., a non-cervical cancer sample, orbetween samples from different sources.

[0279] Alternatively, the expression level can be provided as a relativeexpression level. To determine a relative expression level of a marker,the level of expression of the marker is determined for 10 or moresamples of normal versus cancer cell isolates, preferably 50 or moresamples, prior to the determination of the expression level for thesample in question. The mean expression level of each of the genesassayed in the larger number of samples is determined and this is usedas a baseline expression level for the marker. The expression level ofthe marker determined for the test sample (absolute level of expression)is then divided by the mean expression value obtained for that marker.This provides a relative expression level.

[0280] Preferably, the samples used in the baseline determination willbe from cervical cancer or from non-cervical cancer cells of cervicaltissue. The choice of the cell source is dependent on the use of therelative expression level. Using expression found in normal tissues as amean expression score aids in validating whether the marker assayed iscervical specific (versus normal cells). In addition, as more data isaccumulated, the mean expression value can be revised, providingimproved relative expression values based on accumulated data.Expression data from cervical cells provides a means for grading theseverity of the cervical cancer state.

[0281] In another embodiment of the present invention, a marker proteinis detected. A preferred agent for detecting marker protein of theinvention is an antibody capable of binding to such a protein or afragment thereof, preferably an antibody with a detectable label.Antibodies can be polyclonal, or more preferably, monoclonal. An intactantibody, or a fragment or derivative thereof (e.g., Fab or F(ab′)₂) canbe used. The term “labeled”, with regard to the probe or antibody, isintended to encompass direct labeling of the probe or antibody bycoupling (i.e., physically linking) a detectable substance to the probeor antibody, as well as indirect labeling of the probe or antibody byreactivity with another reagent that is directly labeled. Examples ofindirect labeling include detection of a primary antibody using afluorescently labeled secondary antibody and end-labeling of a DNA probewith biotin such that it can be detected with fluorescently labeledstreptavidin.

[0282] Proteins from cervical cells can be isolated using techniquesthat are well known to those of skill in the art. The protein isolationmethods employed can, for example, be such as those described in Harlowand Lane (Harlow and Lane, 1988, Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).

[0283] A variety of formats can be employed to determine whether asample contains a protein that binds to a given antibody. Examples ofsuch formats include, but are not limited to, enzyme immunoassay (EIA),radioimmunoassay (RIA), Western blot analysis and enzyme linkedimmunoabsorbant assay (ELISA). A skilled artisan can readily adapt knownprotein/antibody detection methods for use in determining whethercervical cells express a marker of the present invention.

[0284] In one format, antibodies, or antibody fragments or derivatives,can be used in methods such as Western blots or immunofluorescencetechniques to detect the expressed proteins. In such uses, it isgenerally preferable to immobilize either the antibody or proteins on asolid support. Suitable solid phase supports or carriers include anysupport capable of binding an antigen or an antibody. Well-knownsupports or carriers include glass, polystyrene, polypropylene,polyethylene, dextran, nylon, amylases, natural and modified celluloses,polyacrylamides, gabbros, and magnetite.

[0285] One skilled in the art will know many other suitable carriers forbinding antibody or antigen, and will be able to adapt such support foruse with the present invention. For example, protein isolated fromcervical cells can be run on a polyacrylamide gel electrophoresis andimmobilized onto a solid phase support such as nitrocellulose. Thesupport can then be washed with suitable buffers followed by treatmentwith the detectably labeled antibody. The solid phase support can thenbe washed with the buffer a second time to remove unbound antibody. Theamount of bound label on the solid support can then be detected byconventional means.

[0286] The invention also encompasses kits for detecting the presence ofa marker protein or nucleic acid in a biological sample (e.g., cervicalsmear). Such kits can be used to determine if a subject is sufferingfrom or is at increased risk of developing cervical cancer. For example,the kit can comprise a labeled compound or agent capable of detecting amarker protein or nucleic acid in a biological sample and means fordetermining the amount of the protein or mRNA in the sample (e.g., anantibody which binds the protein or a fragment thereof, or anoligonucleotide probe which binds to DNA or mRNA encoding the protein).Kits can also include instructions for interpreting the results obtainedusing the kit.

[0287] For antibody-based kits, the kit can comprise, for example: (1) afirst antibody (e.g., attached to a solid support) which binds to amarker protein; and, optionally, (2) a second, different antibody whichbinds to either the protein or the first antibody and is conjugated to adetectable label.

[0288] For oligonucleotide-based kits, the kit can comprise, forexample: (1) an oligonucleotide, e.g., a detectably labeledoligonucleotide, which hybridizes to a nucleic acid sequence encoding amarker protein or (2) a pair of primers useful for amplifying a markernucleic acid molecule. The kit can also comprise, e.g., a bufferingagent, a preservative, or a protein stabilizing agent. The kit canfurther comprise components necessary for detecting the detectable label(e.g., an enzyme or a substrate). The kit can also contain a controlsample or a series of control samples which can be assayed and comparedto the test sample. Each component of the kit can be enclosed within anindividual container and all of the various containers can be within asingle package, along with instructions for interpreting the results ofthe assays performed using the kit.

[0289] B. Pharmacogenomics

[0290] The markers of the invention are also useful as pharmacogenomicmarkers. As used herein, a “pharmacogenomic marker” is an objectivebiochemical marker whose expression level correlates with a specificclinical drug response or susceptibility in a patient (see, e.g., McLeodet al. (1999) Eur. J. Cancer 35(12): 1650-1652). The presence orquantity of the pharmacogenomic marker expression is related to thepredicted response of the patient and more particularly the patient'stumor to therapy with a specific drug or class of drugs. By assessingthe presence or quantity of the expression of one or morepharmacogenomic markers in a patient, a drug therapy which is mostappropriate for the patient, or which is predicted to have a greaterdegree of success, may be selected. For example, based on the presenceor quantity of RNA or protein encoded by specific tumor markers in apatient, a drug or course of treatment may be selected that is optimizedfor the treatment of the specific tumor likely to be present in thepatient. The use of pharmacogenomic markers therefore permits selectingor designing the most appropriate treatment for each cancer patientwithout trying different drugs or regimes.

[0291] Another aspect of pharmacogenomics deals with genetic conditionsthat alters the way the body acts on drugs. These pharmacogeneticconditions can occur either as rare defects or as polymorphisms. Forexample, glucose-6-phosphate dehydrogenase (G6PD) deficiency is a commoninherited enzymopathy in which the main clinical complication ishemolysis after ingestion of oxidant drugs (anti-malarials,sulfonamides, analgesics, nitrofurans) and consumption of fava beans.

[0292] As an illustrative embodiment, the activity of drug metabolizingenzymes is a major determinant of both the intensity and duration ofdrug action. The discovery of genetic polymorphisms of drug metabolizingenzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymesCYP2D6 and CYP2C19) has provided an explanation as to why some patientsdo not obtain the expected drug effects or show exaggerated drugresponse and serious toxicity after taking the standard and safe dose ofa drug. These polymorphisms are expressed in two phenotypes in thepopulation, the extensive metabolizer (EM) and poor metabolizer (PM).The prevalence of PM is different among different populations. Forexample, the gene coding for CYP2D6 is highly polymorphic and severalmutations have been identified in PM, which all lead to the absenceoffinctional CYP2D6. Poormetabolizers of CYP2D6 and CYP2C19 quitefrequently experience exaggerated drug response and side effects whenthey receive standard doses. If a metabolite is the active therapeuticmoiety, a PM will show no therapeutic response, as demonstrated for theanalgesic effect of codeine mediated by its CYP2D6-forned metabolitemorphine. The other extreme are the so called ultra-rapid metabolizerswho do not respond to standard doses. Recently, the molecular basis ofultra-rapid metabolism has been identified to be due to CYP2D6 geneamplification.

[0293] Thus, the level of expression of a marker of the invention in anindividual can be determined to thereby select appropriate agent(s) fortherapeutic or prophylactic treatment of the individual. In addition,pharmacogenetic studies can be used to apply genotyping of polymorphicalleles encoding drug-metabolizing enzymes to the identification of anindividual's drug responsiveness phenotype. This knowledge, when appliedto dosing or drug selection, can avoid adverse reactions or therapeuticfailure and thus enhance therapeutic or prophylactic efficiency whentreating a subject with a modulator of expression of a marker of theinvention.

[0294] C. Monitoring Clinical Trials

[0295] Monitoring the influence of agents (e.g., drug compounds) on thelevel of expression of a marker of the invention can be applied not onlyin basic drug screening, but also in clinical trials. For example, theeffectiveness of an agent to affect marker expression can be monitoredin clinical trials of subjects receiving treatment for cervical cancer.In a preferred embodiment, the present invention provides a method formonitoring the effectiveness of treatment of a subject with an agent(e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleicacid, small molecule, or other drug candidate) comprising the steps of(i) obtaining a pre-administration sample from a subject prior toadministration of the agent; (ii) detecting the level of expression ofone or more selected markers of the invention in the pre-administrationsample; (iii) obtaining one or more post-administration samples from thesubject; (iv) detecting the level of expression of the marker(s) in thepost-administration samples; (v) comparing the level of expression ofthe marker(s) in the pre-administration sample with the level ofexpression of the marker(s) in the post-administration sample orsamples; and (vi) altering the administration of the agent to thesubject accordingly. For example, increased expression of the markergene(s) during the course of treatment may indicate ineffective dosageand the desirability of increasing the dosage. Conversely, decreasedexpression of the marker gene(s) may indicate efficacious treatment andno need to change dosage.

[0296] D. Electronic Apparatus Readable Media and Arrays

[0297] Electronic apparatus readable media comprising a marker of thepresent invention is also provided. As used herein, “electronicapparatus readable media” refers to any suitable medium for storing,holding or containing data or information that can be read and accesseddirectly by an electronic apparatus. Such media can include, but are notlimited to: magnetic storage media, such as floppy discs, hard discstorage medium, and magnetic tape; optical storage media such as compactdisc; electronic storage media such as RAM, ROM, EPROM, EEPROM and thelike; general hard disks and hybrids of these categories such asmagnetic/optical storage media. The medium is adapted or configured forhaving recorded thereon a marker of the present invention.

[0298] As used herein, the term “electronic apparatus” is intended toinclude any suitable computing or processing apparatus or other deviceconfigured or adapted for storing data or information. Examples ofelectronic apparatus suitable for use with the present invention includestand-alone computing apparatus; networks, including a local areanetwork (LAN), a wide area network (WAN) Internet, Intranet, andExtranet; electronic appliances such as a personal digital assistants(PDAs), cellular phone, pager and the like; and local and distributedprocessing systems.

[0299] As used herein, “recorded” refers to a process for storing orencoding information on the electronic apparatus readable medium. Thoseskilled in the art can readily adopt any of the presently known methodsfor recording information on known media to generate manufacturescomprising the markers of the present invention.

[0300] A variety of software programs and formats can be used to storethe marker information of the present invention on the electronicapparatus readable medium. For example, the marker nucleic acid sequencecan be represented in a word processing text file, formatted incommercially-available software such as WordPerfect and MicroSoft Word,or represented in the form of an ASCII file, stored in a databaseapplication, such as DB2, Sybase, Oracle, or the like, as well as inother forms. Any number of data processor structuring formats (e.g.,text file or database) may be employed in order to obtain or create amedium having recorded thereon the markers of the present invention.

[0301] By providing the markers of the invention in readable form, onecan routinely access the marker sequence information for a variety ofpurposes. For example, one skilled in the art can use the nucleotide oramino acid sequences of the present invention in readable form tocompare a target sequence or target structural motif with the sequenceinformation stored within the data storage means. Search means are usedto identify fragments or regions of the sequences of the invention whichmatch a particular target sequence or target motif.

[0302] The present invention therefore provides a medium for holdinginstructions for performing a method for determining whether a subjecthas cervical cancer or a pre-disposition to cervical cancer, wherein themethod comprises the steps of determining the presence or absence of amarker and based on the presence or absence of the marker, determiningwhether the subject has cervical cancer or a pre-disposition to cervicalcancer and/or recommending a particular treatment for cervical cancer orpre-cervical cancer condition.

[0303] The present invention further provides in an electronic systemand/or in a network, a method for determining whether a subject hascervical cancer or a pre-disposition to cervical cancer associated witha marker wherein the method comprises the steps of determining thepresence or absence of the marker, and based on the presence or absenceof the marker, determining whether the subject has cervical cancer or apre-disposition to cervical cancer, and/or recommending a particulartreatment for the cervical cancer or pre-cervical cancer condition. Themethod may further comprise the step of receiving phenotypic informationassociated with the subject and/or acquiring from a network phenotypicinformation associated with the subject.

[0304] The present invention also provides in a network, a method fordetermining whether a subject has cervical cancer or a pre-dispositionto cervical cancer associated with a marker, said method comprising thesteps of receiving information associated with the marker receivingphenotypic information associated with the subject, acquiringinformation from the network corresponding to the marker and/or cervicalcancer, and based on one or more of the phenotypic information, themarker, and the acquired information, determining whether the subjecthas a cervical cancer or a pre-disposition to cervical cancer. Themethod may further comprise the step of recommending a particulartreatment for the cervical cancer or pre-cervical cancer condition.

[0305] The present invention also provides a business method fordetermining whether a subject has cervical cancer or a pre-dispositionto cervical cancer, said method comprising the steps of receivinginformation associated with the marker, receiving phenotypic informationassociated with the subject, acquiring information from the networkcorresponding to the marker and/or cervical cancer, and based on one ormore of the phenotypic information, the marker, and the acquiredinformation, determining whether the subject has cervical cancer or apre-disposition to cervical cancer. The method may further comprise thestep of recommending a particular treatment for the cervical cancer orpre-cervical cancer condition.

[0306] The invention also includes an array comprising a marker of thepresent invention. The array can be used to assay expression of one ormore genes in the array. In one embodiment, the array can be used toassay gene expression in a tissue to ascertain tissue specificity ofgenes in the array. In this manner, up to about 7600 genes can besimultaneously assayed for expression. This allows a profile to bedeveloped showing a battery of genes specifically expressed in one ormore tissues.

[0307] In addition to such qualitative determination, the inventionallows the quantitation of gene expression. Thus, not only tissuespecificity, but also the level of expression of a battery of genes inthe tissue is ascertainable. Thus, genes can be grouped on the basis oftheir tissue expression per se and level of expression in that tissue.This is useful, for example, in ascertaining the relationship of geneexpression between or among tissues. Thus, one tissue can be perturbedand the effect on gene expression in a second tissue can be determined.In this context, the effect of one cell type on another cell type inresponse to a biological stimulus can be determined. Such adetermination is useful, for example, to know the effect of cell-cellinteraction at the level of gene expression. If an agent is administeredtherapeutically to treat one cell type but has an undesirable effect onanother cell type, the invention provides an assay to determine themolecular basis of the undesirable effect and thus provides theopportunity to co-administer a counteracting agent or otherwise treatthe undesired effect. Similarly, even within a single cell type,undesirable biological effects can be determined at the molecular level.Thus, the effects of an agent on expression of other than the targetgene can be ascertained and counteracted.

[0308] In another embodiment, the array can be used to monitor the timecourse of expression of one or more genes in the array. This can occurin various biological contexts, as disclosed herein, for exampledevelopment of cervical cancer, progression of cervical cancer, andprocesses, such a cellular transformation associated with cervicalcancer.

[0309] The array is also useful for ascertaining the effect of theexpression of a gene on the expression of other genes in the same cellor in different cells. This provides, for example, for a selection ofalternate molecular targets for therapeutic intervention if the ultimateor downstream target cannot be regulated.

[0310] The array is also useful for ascertaining differential expressionpatterns of one or more genes in normal and abnormal cells. Thisprovides a battery of genes that could serve as a molecular target fordiagnosis or therapeutic intervention.

[0311] E. Surrogate Markers

[0312] The markers of the invention may serve as surrogate markers forone or more disorders or disease states or for conditions leading up todisease states, and in particular, cervical cancer. As used herein, a“surrogate marker” is an objective biochemical marker which correlateswith the absence or presence of a disease or disorder, or with theprogression of a disease or disorder (e.g., with the presence or absenceof a tumor). The presence or quantity of such markers is independent ofthe disease. Therefore, these markers may serve to indicate whether aparticular course of treatment is effective in lessening a disease stateor disorder. Surrogate markers are of particular use when the presenceor extent of a disease state or disorder is difficult to assess throughstandard methodologies (e.g., early stage tumors), or when an assessmentof disease progression is desired before a potentially dangerousclinical endpoint is reached (e.g., an assessment of cardiovasculardisease may be made using cholesterol levels as a surrogate marker, andan analysis of HIV infection may be made using HIV RNA levels as asurrogate marker, well in advance of the undesirable clinical outcomesof myocardial infarction or fully-developed AIDS). Examples of the useof surrogate markers in the art include: Koomen et al. (2000) J. Mass.Spectrom. 35: 258-264; and James (1994) AIDS Treatment News Archive 209.

[0313] The markers of the invention are also useful as pharmacodynamicmarkers. As used herein, a “pharmacodynamic marker” is an objectivebiochemical marker which correlates specifically with drug effects. Thepresence or quantity of a pharmacodynamic marker is not related to thedisease state or disorder for which the drug is being administered;therefore, the presence or quantity of the marker is indicative of thepresence or activity of the drug in a subject. For example, apharmacodynamic marker may be indicative of the concentration of thedrug in a biological tissue, in that the marker is either expressed ortranscribed or not expressed or transcribed in that tissue inrelationship to the level of the drug. In this fashion, the distributionor uptake of the drug may be monitored by the pharmacodynamic marker.Similarly, the presence or quantity of the pharmacodynamic marker may berelated to the presence or quantity of the metabolic product of a drug,such that the presence or quantity of the marker is indicative of therelative breakdown rate of the drug in vivo. Pharmacodynamic markers areof particular use in increasing the sensitivity of detection of drugeffects, particularly when the drug is administered in low doses. Sinceeven a small amount of a drug may be sufficient to activate multiplerounds of marker transcription or expression, the amplified marker maybe in a quantity which is more readily detectable than the drug itself.Also, the marker may be more easily detected due to the nature of themarker itself; for example, using the methods described herein,antibodies may be employed in an immune-based detection system for aprotein marker, or marker-specific radiolabeled probes may be used todetect a mRNA marker. Furthermore, the use of a pharmacodynamic markermay offer mechanism-based prediction of risk due to drug treatmentbeyond the range of possible direct observations. Examples of the use ofpharmacodynamic markers in the art include: Matsuda et al. U.S. Pat. No.6,033,862; Hattis et aL (1991) Env. Health Perspect. 90: 229-238;Schentag (1999) Am. J Health-Syst. Pharm. 56 Suppl. 3: S21-S24; andNicolau (1999) Am, J. Health-Syst. Pharm. 56 Suppl. 3: S16-S20.

[0314] VI. Experimental Protocol

[0315] A. Identification of Clones

[0316] Cervical tumor specific cDNA clones were identified bytranscription profiling using mRNA from 12 cervical tumors, 5 CIN III, 5CIN I and 12 normal cervical tissues. The subtracted libraries wereconstructed using mRNA from at least three independent normalectocervix, B-lymphocytes, T-lymphocytes and other white blood cells (inactivated and resting states) as drivers and four independent stage 1Bcervical tumors or four independent C1N III cervical samples as testers.The top up-regulated clones in tumors or C1N III cervical tissues, asdetermined by proprietary statistical analysis methods, were selected.The clusters in which the selected clones belong were blasted againstboth public and proprietary sequence databases in order to identifyother EST sequences or clusters with significant overlap. Thus,contiguous EST sequences and/or clusters were assembled into full-lengthgenes.

[0317] An identification of protein sequence corresponding to the clonewas accomplished by obtaining one of the following:

[0318] a) a direct match between the protein sequence and at least oneEST sequence in one of its 6 possible translations;

[0319] b) a direct match between the nucleotide sequence for the mRNAcorresponding to the protein sequence and at least one EST sequence;

[0320] c) a match between the protein sequence and a contiguous assembly(contig) of the EST sequences with other available EST sequences in thedatabases in one of its 6 possible translations; or

[0321] d) a match between the nucleotide sequence for the mRNAcorresponding to the protein sequence and a contiguous assembly of theEST sequences with other available EST sequences in the databases in oneof its 6 possible translations.

[0322] VII. Summary of the Data

[0323] Tables 1-3 list the markers obtained using the foregoingprotocol. The tables provide the name of the gene corresponding to themarker (“Gene Name”), the sequence listing identifier of the cDNAsequence of a nucleotide transcript encoded by or corresponding to themarker (“SEQ ID NO (nts)”), the sequence listing identifier of the aminoacid sequence of a protein encoded by the nucleotide transcript (“SEQ IDNO (AAs)”), and the location of the protein coding sequence within thecDNA sequence (“CDS”).

[0324] Table 1 lists all of the markers of the invention which areover-expressed in cervical cancer cells compared to normal (i.e.,non-cancerous) cervical cells. Table 2 lists newly-identified nucleotideand amino acid sequences useful as cervical cancer markers. Table 3lists newly-identified nucleotide sequences useful as cervical cancermarkers.

[0325] Other Embodiments

[0326] Those skilled in the art will recognize, or be able to ascertainusing no more than routine experimentation, many equivalents to thespecific embodiments of the invention described herein. Such equivalentsare intended to be encompassed by the following claims:

0 SEQUENCE LISTING The patent application contains a lengthy “SequenceListing” section. A copy of the “Sequence Listing” is available inelectronic form from the USPTO web site(http://seqdata.uspto.gov/sequence.html?DocID=20030087270). Anelectronic copy of the “Sequence Listing” will also be available fromthe USPTO upon request and payment of the fee set forth in 37 CFR1.19(b)(3).

What is claimed:
 1. An isolated nucleic acid molecule comprising anucleotide sequence selected from the group consisting of SEQ ID NOs: 1,3, 5, 7, 143, 145, 147, 149, 151, 167, 203, 217, 231, 233, 51, 65, 67,68, 100, and
 153. 2. A vector which contains the nucleic acid moleculeof claim
 1. 3. A host cell which contains the nucleic acid molecule ofclaim
 1. 4. A method of assessing whether a patient is afflicted withcervical cancer, the method comprising comparing: a) the level ofexpression of a marker in a patient sample, wherein the marker isselected from Table 1; and b) the normal level of expression of themarker in a control non-cervical cancer sample, wherein a significantincrease in the level of expression of the marker in the patient sampleand the normal level is an indication that the patient is afflicted withcervical cancer.
 5. An isolated polypeptide which is encoded by anucleic acid molecule comprising a nucleotide sequence selected from thegroup consisting of SEQ ID NOs: 1, 3, 5, 7, 143, 145, 147, 149, 151,167, 203, 217, 231, and
 233. 6. An antibody which selectively binds tothe polypeptide of claim
 5. 7. An isolated polypeptide comprising anamino acid sequence selected from the group consisting of SEQ ID NOs: 2,4, 6, 8, 144, 146, 148, 150, 152, 168, 204, 218, 232, and
 234. 8. Anantibody which selectively binds to the polypeptide of claim 7.